Methods of treatment using IL-18 receptor antigen binding proteins

ABSTRACT

Provided herein are IL-18 receptor antigen binding proteins and polynucleotides encoding the same. Expression vectors and host cells comprising the same for production of the antigen binding proteins are also provided. In addition, provided are compositions and methods for diagnosing and treating diseases mediated by IL-18 receptor.

This application is a divisional of U.S. patent application Ser. No.12/670,112, filed Aug. 16, 2010, now U.S. Pat. No. 8,257,707 which is aNational Stage application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2008/071047 (which designated the United States),having an international filing date of Jul. 24, 2008, which claims thepriority benefit of U.S. Provisional Patent Application Ser. No.61,073,142, filed Jun. 17, 2008, U.S. Provisional Patent ApplicationSer. No. 60/951,692, filed Jul. 24, 2007, and U.S. Provisional PatentApplication Ser. No. 60/951,691, filed Jul. 24, 2007 each of which ishereby incorporated by reference in its entirety.

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequence_Listing.txt, created Aug. 2, 2012, which is 135 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

I. FIELD OF THE INVENTION

Provided herein are IL-18 receptor antigen binding proteins andpolynucleotides encoding the same. Expression vectors and host cellscomprising the same for production of the antigen binding proteins arealso provided. In addition, provided are compositions and methods fordiagnosing and treating diseases mediated by IL-18 receptor.

II. BACKGROUND

IL-18 is a proinflammatory cytokine that belongs to the IL-1 family ofligands. Okamura et al., 1995, Nature 378:88-91. Also referred to asIFN-γ-inducing factor, IL-18 is a cytokine that plays an important rolein the TH1 response, primarily by its ability to induce IFN-γ productionin T cells and natural killer cells. IL-18 is related to the IL-1 familyin both structure and function. In terms of structure, IL-18 and IL-1βshare significant primary amino acid sequences and are both folded asβ-sheet polypeptides. In terms of function, IL-18 induces geneexpression and synthesis of IL-1, TNF, Fas ligand, and severalcytokines.

The activity of IL-18 is transduced through a signal transducing pathwayinitiated by its forming of a IL-18 receptor (IL-18R) complex. TheIL-18R includes a binding chain termed α-IL-18 receptor (α-IL-18R), amember of the IL-IR family previously identified as the IL-1R-relatedprotein (IL-1Rrp), and a β-IL-18 receptor (β-IL-18R), also a member ofthe IL-1R family and previously identified as AcPL; both chains arerequired for signaling. Born et al., 1998, J. Biol. Chem. 273:29445-50.The IL-18/IL-18R complex recruits IL-1R-activating kinase and TNFreceptor-associated factor-6, which phosphorylates nuclear factor kappaB(NFkappaB)-inducing kinase with subsequent activation of NFkappaB. IL-18participates in both innate and acquired immunity. Dinarello, 1999, J.Allergy Clin. Immun. 103:11-24.

Increased levels of IL-18 and/or involvement of IL-18 mediated signalsin pathogenesis have been demonstrated in a variety of human diseasestates, including autoimmune diseases (WO2004/002519; WO2005/063290;WO2004/034988; Mallat et al., 2002, Circ. Res. 91:441-448), hepaticdiseases (Finitto et al., 2004, Liver 53:392-400; Tsutsui et al., 2000,Immunological Reviews 174:192-209; Ludwiczek et al., 2002, J. ClinicalImmunology 22:331-337), pancreatic diseases, and cardiovascular diseases(Gerdes et al, 2002, J. Exp. Med. 195:245-257; WO03/080104; WO02/060479;WO01/85201; Raeburn et al., 2002, Am. J. Physiol. Heart Circ. Physiol.283:H650-H657). Accordingly, it is desirable to generate new agentscapable of modulating the IL-18/IL-18 receptor interaction.

III. SUMMARY

Provided herein are α- and β-IL-18 receptor (also referred to hereincollectively as “IL-18 receptor” or “IL-18R”) antigen binding proteinsand polynucleotides that encode them. The IL-18 receptor antigen bindingproteins inhibit, interfere with, or modulate at least one of thebiological responses mediated by IL-18 and as such can be useful forameliorating the effects of IL-18 mediated diseases or disorders. Alsoprovided are expression systems, including cell lines, for theproduction of α- and β-IL-18 receptor antigen binding proteins andmethods for diagnosing and treating diseases associated with aberrantIL-18 activity.

In one embodiment, antigen binding proteins bind the α- and β-IL-18receptor, and comprise (a) a scaffold structure; and (b) at least onecomplementary determining region (CDR), selected from the CDRH regionsof any of SEQ ID NOs:89-139 or the CDRL regions of any of SEQ IDNOs:140-190. In this embodiment, of particular use are antigen bindingproteins with a CDRH3 or CDRL3 region of SEQ ID NO:91, 94, 97, 100, 103,106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139 or SEQ IDNO:142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181,184, 187, 190, respectively. Additional embodiments utilize antigenbinding proteins with one CDR selected from the CDRH regions of any ofSEQ ID NOs:89-139 and a CDRL region of any of SEQ ID NOs:140-190 (e.g.,the antigen binding protein has two CDR regions, one heavy and onelight; again, in a specific embodiment the antigen binding proteins haveboth a CDRH3 and a CDRL3 region).

The antigen binding proteins can bind to an IL-18 receptor α- or β-chainhaving the amino acid sequence of SEQ ID NO:69 or SEQ ID NO:71,respectively.

Described herein are antigen binding proteins that comprise a heavychain amino acid sequence that comprises at least one CDR selected fromthe group consisting of: (a) a CDRH1 of any of SEQ ID NOs:89, 92, 95,98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137; (b)a CDRH2 of any of SEQ ID NOs:90, 93, 96, 99, 102, 105, 108, 111, 114,117, 120, 123, 126, 129, 132, 135, 138; and (c) a CDRH3 of any of SEQ IDNOs:91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130,133, 136, 139; and/or a light chain amino acid sequence that comprisesat least one CDR selected from the group consisting of: (a) a CDRL1 ofany of SEQ ID NOs:140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170,173, 176, 179, 182, 185, 188; (b) a CDRL2 of any of SEQ ID NOs:141, 144,147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186,189; and (c) a CDRL3 of any of SEQ ID NOs:142, 145, 148, 151, 154, 157,160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190.

In certain aspects, the antigen binding protein comprises a heavy chainamino acid sequence having a CDRH1, a CDRH2, and a CDRH3 of any of SEQID NOs:89-139, and/or a light chain amino acid sequence that comprises aCDRL1, a CDRL2, and a CDRL3 of any of SEQ ID NOs:140-190. Preferredantigen binding proteins comprise a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOs:1-17 and/or a lightchain amino acid sequence selected from the group consisting of SEQ IDNOs:18-34. Preferred CDRH3s include those set forth in any of SEQ IDNOs:91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130,133, 136, 139. Preferred CDRL3s include those set forth in any of SEQ IDNOs:142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178,181, 184, 187, 190.

In certain aspects, the antigen binding protein comprises one or moreIgG heavy or light chains, including those of the IgG1-, IgG2- IgG3- orIgG4-type. Preferred IgG heavy chains include, but are not limited to,those set forth in SEQ ID NO:73, 77, 81, and 85. Preferred IgG lightchains include, but are not limited to, those set forth in SEQ ID NO:75,79, 83, and 87.

As described herein, antigen binding proteins that bind to amino acidresidues 250-253 and 267-271 of a three dimensional structure formed bymature α-IL-18 receptor (SEQ ID NO:69) are particularly useful inblocking the interaction of IL-18 with IL-18 receptor.

An antigen binding protein can be a monoclonal antibody, a humanantibody, a recombinant antibody, a chimeric antibody, a humanizedantibody, a bispecific antibody, or a fragment thereof. Antibodyfragments include, but are not limited to, a minibody, a domainantibody, a F(ab) fragment, a F(ab′) fragment, a F(ab′)₂ fragment, a Fvfragment, a scFv fragment, a Fd fragment, a diabody, or a single chainantibody molecule.

In other aspects, provided herein are isolated nucleic acids encodingone or more IL-18 receptor antigen binding proteins. Such nucleic acidscan be comprised within a vector and operably linked to a controlsequence. Also, provided herein are host cells transformed with suchisolated nucleic acids.

Additionally, provided herein are pharmaceutical compositions comprisingan IL-18 receptor antigen binding protein and a pharmaceuticallyacceptable carrier. Such pharmaceutical compositions are useful inmethods for preventing or treating a condition associated with IL-18receptor in a patient, which comprise administering an effective amountthereof to the patient. Diseases and conditions associated with IL-18receptor include inflammatory and autoimmmune diseases (such aspsoriasis, rheumatoid arthritis, juvenile idiopathic arthritis, Still'sdisease, ankylosing spondylitis, osteo arthritis, ulcerative arthritis,coleliac disease, psoriatic arthritis, chronic obstructive pulmonarydisease, asthma, particularly chronic severe asthma, acute respiratorydistress syndrome, sepsis, Alzheimer disease, lupus, allergic rhinitis,idiopathic thrombocytopenic purpura, transplantation, atopic dermatitis,type II diabetes, Crohn's disease, inflammatory bowel disease, multiplesclerosis, autoimmune hepatitis, HIV, atopic dermatitis, myastheniagravis, sarcoidosis), a hepatic disease (such as hepatitis), apancreatic disease (such as chronic pancreatitis or acute pancreatitis),and a cardiovascular disease (such as acute heart attacks, atheromatousplaque rupture, post-ischemic heart failure, reperfusion injury,vascular inflammation, chronic heart failure, artherosclerosis,cardiovascular complications of rheumatoid arthritis, andatherogenesis).

Further provided herein are methods of inhibiting the binding of IL-18to IL-18 receptor comprising contacting an IL-18 receptor with an IL-18receptor antigen binding protein. Upon binding IL-18 receptor, the IL-18receptor antigen binding protein will prevent or block binding of thereceptor to IL-18.

IV. DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1O, 1P,and 1Q depict nucleic acid and amino acid sequences of VH and VLvariable domains of α- and β-IL-18 receptor antigen binding proteins.

FIGS. 2A, 2B, 2C, 2D, and 2E show the CDR1, CDR2, and CDR3 regions ofvarious heavy and light chain variable regions of antigen bindingproteins. The amino acid sequences of the various heavy and light chainregions are identified in SEQ ID NOs:1-34. The sequences of theindividual CDRs are identified in SEQ ID NOs:89-190.

FIGS. 3A and 3B depict an alignment of the amino acid sequences of heavyand light chain variable sequences of α- and β-IL-18 receptor antigenbinding proteins. The CDR1, CDR2 and CDR3 regions are highlighted ingrey.

FIG. 4 depicts a chart showing various possible combinations of heavychain variable regions and light chain variable region sequences. Shownare dimers of each one heavy and one light chain variable region. Asnaturally occurring antibodies typically are tetramers, an antibody maycomprise a combination of two of the depicted dimers.

FIG. 5 depicts the portions of the α-IL-18 receptor amino acid sequencesthat form the epitope for a specific antigen binding protein embodiment.

FIG. 6 depicts the complete AM_(H)6 heavy chain nucleotide and aminoacid sequences (SEQ ID NOs:74 and 73, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 7 depicts the complete AM_(L)12 light chain nucleotide and aminoacid sequences (SEQ ID NOs:76 and 75, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 8 depicts the complete AM_(H)4 heavy chain nucleotide and aminoacid sequences (SEQ ID NOs:78 and 77, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 9 depicts the complete AM_(L)14 light chain nucleotide and aminoacid sequences (SEQ ID NOs:80 and 79, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 10 depicts the complete AM_(H)9 heavy chain nucleotide and aminoacid sequences (SEQ ID NOs:82 and 81, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 11 depicts the complete AM_(L)9 light chain nucleotide and aminoacid sequences (SEQ ID NOs:84 and 83, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 12 depicts the complete AM_(H)11 heavy chain nucleotide and aminoacid sequences (SEQ ID NOs:86 and 85, respectively). The arrow indicatesthe cleavage site of the leader sequence.

FIG. 13 depicts the complete AM_(L)7 light chain nucleotide and aminoacid sequences (SEQ ID NOs:88 and 87, respectively). The arrow indicatesthe cleavage site of the leader sequence.

V. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques may be used for chemicalsynthesis, chemical analyses, pharmaceutical preparation, formulation,and delivery and treatment of patients.

A. General Overview

Provided herein are antigen binding proteins that bind an α- or β-IL-18receptor; the amino acid sequence of the human α- and β-IL-18 receptorare depicted in SEQ ID NOs:69 and 71, respectively. The antigen bindingproteins of the invention comprise a scaffold structure with one or morecomplementarity-determining region (CDRs) as depicted in FIGS. 2A-2E, 3Aand 3B, namely the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 portionof SEQ ID NOs:1-34 (see, also SEQ ID NOs:89-292, depicting the aminoacid sequences of the various CDRs). In certain embodiments, thescaffold structure of the antigen binding proteins is based onantibodies (including, but not limited to, monoclonal antibodies, humanantibodies, murine antibodies, chimeric antibodies, humanizedantibodies, bispecific antibodies), antibody fragments (such asminibodies, domain antibodies, F(ab) fragments, F(ab′) fragments, F(ab)₂fragments, F(ab′)₂ fragments, Fv fragments, scFv fragments, Fdfragments), synthetic antibodies (sometimes referred to herein as“antibody mimetics”), antibody fusions (sometimes referred to as“antibody conjugates”), including Fc fusions. The various structures arefurther described and defined hereinbelow.

α- and β-IL-18 receptor antigen binding proteins are useful in treatingconditions associated with IL-18 activity, including TH1-drivenautoimmune diseases (WO2004/002519; WO2005/063290; WO2004/034988; Mallatet al., 2002, Circ. Res. 91:441-448), hepatic diseases (Finitto et al.,2004, Liver 53:392-400; Tsutsui et al., 2000, Immunological Reviews174:192-209; Ludwiczek et al., 2002, J. Clinical Immunology 22:331-337),pancreatic diseases (Yoshida et al., 1998, Anticancer Res. 18:333-5),and cardiovascular diseases (Gerdes et al, 2002, J. Exp. Med.195:245-257; WO03/080104; WO02/060479; WO01/85201; Raeburn et al., 2002,Am. J. Physiol. Heart Circ. Physiol. 283:H650-H657), as is furtherdescribed below. Other uses for antigen binding proteins include, forexample, diagnosis of IL-18 associated diseases or conditions andscreening assays to determine the presence or absence of the α- orβ-IL-18 receptor. Also provided are α- or β-IL-18 receptor antigenbinding proteins, particularly antigen binding proteins that include atleast one complementarity determining region (CDR) including heavyand/or light CDRs, as more fully described below, and combinationsthereof.

The antigen binding proteins of the invention interfere with, block ormodulate the interaction between IL-18 and the IL-18 receptor. In someembodiments, the antigen binding proteins interrupt the IL-18 pathway,thereby decreasing at least one biological activity of IL-18, including,but not limited to, induction of IFN-γ production, induction of killercell formation, and enhancement of cytotoxicity of killer cells. Asdemonstrated in the Examples herein, antigen binding proteins thatreduce IL-18 induced production of IFN-γ by KG cells include thosecomprising AM_(H)8 and AM_(L)11, AM_(H)9 and AM_(L)9, AM_(H)10 andAM_(L)8, AM_(H)11 and AM_(L)7, AM_(H)15 and AM_(L)3, AM_(H)13 andAM_(L)4, AM_(H)13 and AM_(L)5, AM_(H)16 and AM_(L)2, AM_(H)2 andAM_(L)16, AM_(H)2 and AM_(L)17, AM_(H)1 and AM_(L)16, AM_(H)1 andAM_(L)17, AM_(H)4 and AM_(L)14, AM_(H)4 and AM_(L)15, AM_(H)3 andAM_(L)14, AM_(H)3 and AM_(L)15, AM_(H)6 and AM_(L)12, AM_(H)6 andAM_(L)13, AM_(H)5 and AM_(L)12, and AM_(H)5 and AM_(L)13.

The antigen binding proteins of the invention thus may serve to identifyconditions related to IL-18 or IL-18 receptor induced immune responses.In addition, the antigen binding proteins may be utilized to regulateand/or suppress IL-18 or IL-18 receptor mediated immune responses, assuch having efficacy in the treatment and prevention of various diseasescaused by excessive immune responses, e.g., inflammatory diseases.Accordingly, the α- and β-IL-18 receptor antigen binding proteins of thepresent invention can be used for the diagnosis, prevention or treatmentof diseases or conditions associated with the IL-18 and IL-18 receptormediated signal transduction pathway.

B. IL-18 Receptor Antigen Binding Proteins

In one aspect, antigen binding proteins that bind an α- or β-IL-18receptor are provided. By “antigen binding protein” as used herein ismeant a protein that specifically binds a specified antigen. In specificembodiments, the antigen is a human α- or β-IL-18 receptor.

By “protein,” as used herein, is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells,as outlined below. Alternatively, the protein may include syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine), or peptidomimetic structures, i.e., “peptide or proteinanalogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad.Sci. U.S.A. 89:9367, incorporated by reference herein), which can beresistant to proteases or other physiological and/or storage conditions.Such synthetic amino acids may be incorporated in particular when theantigen binding protein is synthesized in vitro by conventional methodswell known in the art. In addition, any combination of peptidomimetic,synthetic and naturally occurring residues/structures can be used.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” may be ineither the (L)- or the (D)-configuration. In a specific embodiment, theamino acids are in the (L)- or (D)-configuration.

In certain aspects, the invention provides recombinant antigen bindingproteins that bind an IL-18 receptor, in some embodiments a human IL-18receptor. In this context, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as described herein. Methods and techniques forthe production of recombinant proteins are well known in the art.

In some embodiments, the antigen binding proteins are isolated proteinsor substantially pure proteins. An “isolated” protein is unaccompaniedby at least some of the material with which it is normally associated inits natural state, preferably constituting at least about 5%, morepreferably at least about 50% by weight of the total protein in a givensample. A “substantially pure” protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes the production of an antigen binding protein from one organismin a different organism or host cell. Alternatively, the protein may bemade at a significantly higher concentration than is normally seen,through the use of an inducible promoter or high expression promoter,such that the protein is made at increased concentration levels.

The antigen binding proteins can specifically bind to an IL-18 receptor,preferably a human IL-18 receptor. “Specifically binds” as used hereinmeans the equilibrium dissociation constant is at least 10⁻⁶ M,preferably 10⁻⁷ to 10⁻¹⁰ M, more preferably <10⁻⁸ to <10⁻¹⁰ M, even morepreferably <10⁻⁹ to <10⁻¹⁰ M. In a specific embodiment, the antigenbinding protein binds to a human IL-18 receptor having the amino acidsequence of SEQ ID NO:69 or 71. An epitope in the α- or β-IL-18 receptorto which preferred antigen binding proteins specifically bind isdetailed below.

In embodiments where the antigen binding protein is used for therapeuticapplications, an important characteristic of an IL-18 receptor antigenbinding protein is whether it can inhibit, interfere with or modulateone or more biological activities of an IL-18 receptor. In this case, anantigen binding protein binds specifically and/or substantially inhibitsbinding of IL-18 to its receptor when an excess of antibody reduces thequantity of IL-18 bound to IL-18 receptor, or vice versa, by at leastabout 20%, 40%, 60%, 80%, 85%, or more (for example by measuring bindingin an in vitro competitive binding assay). IL-18 receptor has manydistinct biological effects, which can be measured in many differentassays in different cell types. The ability of an IL-18 receptor antigenbinding protein to inhibit, interfere with, or modulate the biologicalactivity of IL-18 can be determined, for example, by measuring theinhibition of IFN-γ release in KG1 cells, as described in Example 4 orusing a similar assay in which the ability of an antigen binding proteinto inhibit IFN-γ release is measured.

Not every antigen binding protein that specifically binds to an antigencan block antigen binding to its normal ligand and thus inhibit ormodulate the biological effects of the antigen. As is known in the art,such an effect can depend on what portion of the antigen the antigenbinding protein binds to, and on both the absolute and the relativeconcentrations of the antigen and the antigen binding protein, in thiscase, an IL-18 receptor and the IL-18 receptor antigen binding protein.To be considered capable of inhibiting or modulating the biologicalactivity of an IL-18 receptor as meant herein, an antigen bindingprotein may be able, for example, to inhibit the release of IFN-γobserved in the presence of IL-18, as measured in the KG1 cell assay ofExample 4 or a similar assay, by at least about 20%, 40%, 60%, 80%, 85%,90%, 95%, 99%, or more when the IL-18 concentration is within a range,for example, at about EC₈₀ or EC₉₀, where the effects of an agent thatinhibits its biological activity can be readily apparent. An EC₈₀, asmeant herein, is the amount of IL-18 required for 80% of the maximaleffect of IL-18 to be observed. If the IL-18 concentration is well aboveEC₉₀, effects of an inhibiting agent may be less apparent due to theexcess of IL-18. The concentration of an antigen binding proteinrequired to inhibit, interfere with or modulate the biological activityof IL-18 receptor can vary widely and may depend upon how tightly theantibody binds to IL-18 receptor. For example, one molecule or less ofan antigen binding protein per molecule of IL-18 may be sufficient toinhibit, interfere with or modulate biological activity in the KG1 cellassay. In some embodiments, a ratio of IL-18 receptor/antibody of about1,000:1 to about 1:1,000, including about 2:1, 1:1, 1:2, 1:4, 1:6, 1:8,1:10, 1:20, 1:40, 1:60, 1:100, 1:500, 1:1,000 or more may be required toinhibit, interfere with or modulate the biological activity of IL-18receptor when the IL-18 concentration is from about EC₅₀ to about EC₉₀.Ratios of IL-18 receptor antigen binding protein between these valuesare also possible.

As a general structure, the antigen binding proteins of the inventioncomprise (a) a scaffold, and (b) one or a plurality of CDRs, regionsthat are determinative to antigen binding specificity and affinity. A“complementary determining region” or “CDR,” as used herein, refers to abinding protein region that constitutes the major surface contact pointsfor antigen binding. One or more CDRs are embedded in the scaffoldstructure of the antigen binding protein. The scaffold structure of theantigen binding proteins may be the framework of an antibody, orfragment or variant thereof, or may be completely synthetic in nature.The various scaffold structures of antigen binding proteins are furtherdescribed hereinbelow.

1. CDRs

An antigen binding protein may have six CDRs (as typically does each“arm” of a naturally occurring antibody), for example one heavy chainCDR1 (“CDRH1”), one heavy chain CDR2 (“CDRH2”), one heavy chain CDR3(“CDRH3”), one light chain CDR1 (“CDRL1”), one light chain CDR2(“CDRL2”), one light chain CDR3 (“CDRL3”). The term “naturallyoccurring” as used throughout the specification in connection withbiological materials such as polypeptides, nucleic acids, host cells,and the like, refers to materials which are found in nature. Innaturally occurring antibodies, a CDRH1 typically comprises about five(5) to about seven (7) amino acids, CDRH2 typically comprises aboutsixteen (16) to about nineteen (19) amino acids, and CDRH3 typicallycomprises about three (3) to about twenty five (25) amino acids. CDRL1typically comprises about ten (10) to about seventeen (17) amino acids,CDRL2 typically comprises about seven (7) amino acids, and CDRL3typically comprises about seven (7) to about ten (10) amino acids.Preferred CDRs are depicted in FIGS. 2A-2E, 3A, and 3B.

The structure and properties of CDRs within a naturally occurringantibody are described further in this Section hereinbelow. Briefly, ina traditional antibody scaffold, the CDRs are embedded within aframework in the heavy and light chain variable region where theyconstitute the regions responsible for antigen binding and recognition.A variable region comprises at least three heavy or light chain CDRs,see, supra (Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, Public Health Service N.I.H., Bethesda, Md.; see also Chothiaand Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342: 877-883), within a framework region (designated framework regions1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see alsoChothia and Lesk, 1987, supra). See, infra. The CDRs provided by thepresent invention, however, may not only be used to define the antigenbinding domain of a traditional antibody structure, but may be embeddedin a variety of other scaffold structures, as described herein.

In certain embodiments, one or more CDRs of an antigen binding proteinare each independently selected from the CDRH regions of any of SEQ IDNOs:89-139 and the CDRL regions of any of SEQ ID NOs:140-190. Thus, inone embodiment, the invention provides an antigen binding protein thatbinds an α- or β-IL-18 receptor, wherein said antigen binding proteincomprises (a) a scaffold structure (as described below); and (b) atleast one CDR selected from the CDRH regions of any of SEQ ID NOs:89-139and the CDRL regions of any of SEQ ID NOs:140-190. In this embodiment,of particular use are antigen binding proteins with a CDRH3 or CDRL3region. Additional embodiments utilize antigen binding proteins with oneCDR selected from the CDRH regions of any of SEQ ID NOs:89-139 and aCDRL region of any of SEQ ID NOs:140-190 (e.g., the antigen bindingprotein has two CDR regions, one CDRH and one CDHL, a specificembodiment are antigen binding proteins with both a CDRH3 and a CDRL3region).

As will be appreciated by those in the art, particularly usefulembodiments may contain one, two, three, four, five or six ofindependently selected CDRs of SEQ ID NOs:89-190. However, as will beappreciated by those in the art, specific embodiments generally utilizecombinations of CDRs that are non-repetitive, e.g., antigen bindingproteins are generally not made with two CDRH2 regions, etc.

In some embodiments, antigen binding proteins are generated thatcomprise a CDRH3 region and a CDRL3 region, particularly with the CDRH3region being selected from a CDRH3 region of any of SEQ ID NOs:91, 94,97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139and the CDRL3 region being selected from a CDRL3 region of any of SEQ IDNOs:142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178,181, 184, 187, 190. Particular combinations are depicted in FIG. 4.

In additional embodiments, antigen binding proteins are utilized thatcomprise a CDRH1, a CDRH2, and a CDRH3 region independently selectedfrom SEQ ID NOs:89-139. In more specific embodiments, of particular usemay be antigen binding proteins of this type that have all three CDRHregions selected from the same variable region of any of SEQ IDNOs:1-17.

In further embodiments, antigen binding proteins are utilized thatcomprise a CDRL1, a CDRL2, and a CDRL3 region independently selectedfrom SEQ ID NOs:140-190. In more specific embodiments, of particular useare antigen binding proteins of this type that have all three CDRLregions selected from the same variable region of any of SEQ IDNOs:18-34.

In an additional embodiment, the antigen binding protein comprises aCDRH1, CDRH2, and CDRH3 region independently selected from SEQ IDNOs:89-139, again, in one embodiment with all three regions selectedfrom the same SEQ ID NO, and a CDRL1, CDRL2, and CDRL3 regionindependently selected from SEQ ID NOs:140-190, again, in one embodimentwith all three regions selected from the same variable region of any ofSEQ ID NOs:1-34.

In yet another aspect of the invention provides for an antigen bindingprotein that binds the α- or β IL-18 receptor where the isolated antigenbinding protein comprises a heavy chain amino acid sequence thatcomprises a CDRH1, a CDRH2, or a CDRH3, each selected from any of SEQ IDNOs:89-139, or a fragment thereof, or a light chain amino acid sequencethat comprises a CDRL1, a CDRL2, or a CDRL3, each selected from any ofSEQ ID NOs:140-190, or a fragment thereof. A heavy or light chainvariable region “fragment,” as used herein includes at least one CDR andat least a portion of a framework region of an antibody framework of SEQID NOs:1-34, said portion comprising at least one amino acid.

In yet another aspect, the invention provides for an antigen bindingprotein that binds an α- or β-IL-18 receptor where the isolated antigenbinding protein comprises a heavy chain amino acid sequence thatcomprises a CDRH1, a CDRH2, and a CDRH3, each independently selectedfrom any of SEQ ID NOs:89-139, or a light chain amino acid sequence thatcomprises a CDRL1, a CDRL2, and a CDRL3, each independently selectedfrom any of SEQ ID NOs:140-190. In a specific embodiment, the CDRs arefrom the same contiguous heavy chain amino acid sequence of SEQ IDNOs:1-17 or from the same contiguous light chain amino acid sequence ofSEQ ID NOs:18-34.

A further aspect of the invention provides for an isolated antigenbinding protein that binds an α- or β-IL-18 receptor where the isolatedantigen binding protein comprises a heavy chain amino acid sequence thatcomprises a CDRH1, a CDRH2, and a CDRH3, each independently selectedfrom any of SEQ ID NOs:89-139, and a light chain amino acid sequencethat comprises a CDRL1, a CDRL2, and a CDRL3, each independentlyselected from any of SEQ ID NOs:140-190. In a specific embodiment, theheavy chain CDRs are from the same contiguous heavy chain amino acidsequence of SEQ ID NO:1-17 and the light chain CDRs are from the samecontiguous light chain amino acid sequence of SEQ ID NO:18-34.

An additional aspect of the invention provides for an isolated antigenbinding protein that binds an α- or β-IL-18 receptor where the isolatedantigen binding protein comprises a heavy chain amino acid sequence ofany of SEQ ID NOs:1-17, or a light chain amino acid sequence of any ofSEQ ID NOs:18-34.

A further aspect of the invention provides for an isolated antigenbinding protein that binds an α- or β-IL-18 receptor where the isolatedantigen binding protein comprises a heavy chain amino acid sequence ofany of SEQ ID NOs:1-17, and a light chain amino acid sequence of any ofSEQ ID NOs:18-34. It is noted that the any the heavy chain sequences ofSEQ ID NOs:1-17 can be mixed and matched with any of the light chainsequences of SEQ ID NOs:18-34. The resulting possible combinations aredepicted in FIG. 4. Shown are dimers of a combination of each one heavyand one light chain variable region. As most antibodies are tetramers,an antigen binding protein of the invention may comprise any combinationof any two of the depicted dimers thus including both hetero- andhomo-tetramers, with homo-tetramers (e.g., two identical dimers) beingspecific.

In again a further aspect the antigen binding protein of the inventioncomprises any of the sequences depicted in SEQ ID NOs:73-88.

TABLE 1 provides a brief description of the sequences as they relate totheir sequence identification numbers. The CDRs within the variableregions of the invention are identified in FIGS. 2A-2E, 3A and 3B.

TABLE 1 Brief Description Of Sequence Listings Sequence IdentificationBrief Description Number Amino acid sequence encoding the heavy chainvariable SEQ ID NO: 1 region AM_(H)1 Amino acid sequence encoding theheavy chain variable SEQ ID NO: 2 region AM_(H)2 Amino acid sequenceencoding the heavy chain variable SEQ ID NO: 3 region AM_(H)3 Amino acidsequence encoding the heavy chain variable SEQ ID NO: 4 region AM_(H)4Amino acid sequence encoding the heavy chain variable SEQ ID NO: 5region AM_(H)5 Amino acid sequence encoding the heavy chain variable SEQID NO: 6 region AM_(H)6 Amino acid sequence encoding the heavy chainvariable SEQ ID NO: 7 region AM_(H)7 Amino acid sequence encoding theheavy chain variable SEQ ID NO: 8 region AM_(H)8 Amino acid sequenceencoding the heavy chain variable SEQ ID NO: 9 region AM_(H)9 Amino acidsequence encoding the heavy chain variable SEQ ID NO: 10 region AM_(H)10Amino acid sequence encoding the heavy chain variable SEQ ID NO: 11region AM_(H)11 Amino acid sequence encoding the heavy chain variableSEQ ID NO: 12 region AM_(H)12 Amino acid sequence encoding the heavychain variable SEQ ID NO: 13 region AM_(H)13 Amino acid sequenceencoding the heavy chain variable SEQ ID NO: 14 region AM_(H)14 Aminoacid sequence encoding the heavy chain variable SEQ ID NO: 15 regionAM_(H)15 Amino acid sequence encoding the heavy chain variable SEQ IDNO: 16 region AM_(H)16 Amino acid sequence encoding the heavy chainvariable SEQ ID NO: 17 region AM_(H)17 Amino acid sequence encoding thelight chain variable SEQ ID NO: 18 region AM_(L)1 Amino acid sequenceencoding the light chain variable SEQ ID NO: 19 region AM_(L)2 Aminoacid sequence encoding the light chain variable SEQ ID NO: 20 regionAM_(L)3 Amino acid sequence encoding the light chain variable SEQ ID NO:21 region AM_(L)4 Amino acid sequence encoding the light chain variableSEQ ID NO: 22 region AM_(L)5 Amino acid sequence encoding the lightchain variable SEQ ID NO: 23 region AM_(L)6 Amino acid sequence encodingthe light chain variable SEQ ID NO: 24 region AM_(L)7 Amino acidsequence encoding the light chain variable SEQ ID NO: 25 region AM_(L)8Amino acid sequence encoding the light chain variable SEQ ID NO: 26region AM_(L)9 Amino acid sequence encoding the light chain variable SEQID NO: 27 region AM_(L)10 Amino acid sequence encoding the light chainvariable SEQ ID NO: 28 region AM_(L)11 Amino acid sequence encoding thelight chain variable SEQ ID NO: 29 region AM_(L)12 Amino acid sequenceencoding the light chain variable SEQ ID NO: 30 region AM_(L)13 Aminoacid sequence encoding the light chain variable SEQ ID NO: 31 regionAM_(L)14 Amino acid sequence encoding the light chain variable SEQ IDNO: 32 region AM_(L)15 Amino acid sequence encoding the light chainvariable SEQ ID NO: 33 region AM_(L)16 Amino acid sequence encoding thelight chain variable SEQ ID NO: 34 region AM_(L)17 Nucleotide sequenceencoding the heavy chain variable SEQ ID NO: 35 region AM_(H)1Nucleotide sequence encoding the heavy chain variable SEQ ID NO: 36region AM_(H)2 Nucleotide sequence encoding the heavy chain variable SEQID NO: 37 region AM_(H)3 Nucleotide sequence encoding the heavy chainvariable SEQ ID NO: 38 region AM_(H)4 Nucleotide sequence encoding theheavy chain variable SEQ ID NO: 39 region AM_(H)5 Nucleotide sequenceencoding the heavy chain variable SEQ ID NO: 40 region AM_(H)6Nucleotide sequence encoding the heavy chain variable SEQ ID NO: 41region AM_(H)7 Nucleotide sequence encoding the heavy chain variable SEQID NO: 42 region AM_(H)8 Nucleotide sequence encoding the heavy chainvariable SEQ ID NO: 43 region AM_(H)9 Nucleotide sequence encoding theheavy chain variable SEQ ID NO: 44 region AM_(H)10 Nucleotide sequenceencoding the heavy chain variable SEQ ID NO: 45 region AM_(H)11Nucleotide sequence encoding the heavy chain variable SEQ ID NO: 46region AM_(H)12 Nucleotide sequence encoding the heavy chain variableSEQ ID NO: 47 region AM_(H)13 Nucleotide sequence encoding the heavychain variable SEQ ID NO: 48 region AM_(H)14 Nucleotide sequenceencoding the heavy chain variable SEQ ID NO: 49 region AM_(H)15Nucleotide sequence encoding the heavy chain variable SEQ ID NO: 50region AM_(H)16 Nucleotide sequence encoding the heavy chain variableSEQ ID NO: 51 region AM_(H)17 Nucleotide sequence encoding the lightchain variable SEQ ID NO: 52 region AM_(L)1 Nucleotide sequence encodingthe light chain variable SEQ ID NO: 53 region AM_(L)2 Nucleotidesequence encoding the light chain variable SEQ ID NO: 54 region AM_(L)3Nucleotide sequence encoding the light chain variable SEQ ID NO: 55region AM_(L)4 Nucleotide sequence encoding the light chain variable SEQID NO: 56 region AM_(L)5 Nucleotide sequence encoding the light chainvariable SEQ ID NO: 57 region AM_(L)6 Nucleotide sequence encoding thelight chain variable SEQ ID NO: 58 region AM_(L)7 Nucleotide sequenceencoding the light chain variable SEQ ID NO: 59 region AM_(L)8Nucleotide sequence encoding the light chain variable SEQ ID NO: 60region AM_(L)9 Nucleotide sequence encoding the light chain variable SEQID NO: 61 region AM_(L)10 Nucleotide sequence encoding the light chainvariable SEQ ID NO: 62 region AM_(L)11 Nucleotide sequence encoding thelight chain variable SEQ ID NO: 63 region AM_(L)12 Nucleotide sequenceencoding the light chain variable SEQ ID NO: 64 region AM_(L)13Nucleotide sequence encoding the light chain variable SEQ ID NO: 65region AM_(L)14 Nucleotide sequence encoding the light chain variableSEQ ID NO: 66 region AM_(L)15 Nucleotide sequence encoding the lightchain variable SEQ ID NO: 67 region AM_(L)16 Nucleotide sequenceencoding the light chain variable SEQ ID NO: 68 region AM_(L)17 Aminoacid sequence of human α-IL-18 receptor SEQ ID NO: 69 Nucleotidesequence of human α-IL-18 receptor SEQ ID NO: 70 Amino acid sequence ofhuman β-IL-18 receptor SEQ ID NO: 71 Nucleotide sequence of humanβ-IL-18 receptor SEQ ID NO: 72 Amino acid sequence of complete heavychain of AM_(H)6 SEQ ID NO: 73 Nucleotide sequence of complete heavychain of AM_(H)6 SEQ ID NO: 74 Amino acid sequence of complete lightchain of AM_(L)12 SEQ ID NO: 75 Nucleotide sequence of complete lightchain of AM_(L)12 SEQ ID NO: 76 Amino acid sequence of complete heavychain of AM_(H)4 SEQ ID NO: 77 Nucleotide sequence of complete heavychain of AM_(H)4 SEQ ID NO: 78 Amino acid sequence of complete lightchain of AM_(L)14 SEQ ID NO: 79 Nucleotide sequence of complete lightchain of AM_(L)14 SEQ ID NO: 80 Amino acid sequence of complete heavychain of AM_(H)9 SEQ ID NO: 81 Nucleotide sequence of complete heavychain of AM_(H)9 SEQ ID NO: 82 Amino acid sequence of complete lightchain of AM_(L)9 SEQ ID NO: 83 Nucleotide sequence of complete lightchain of AM_(L)9 SEQ ID NO: 84 Amino acid sequence of complete heavychain of AM_(H)11 SEQ ID NO: 85 Nucleotide sequence of complete heavychain of AM_(H)11 SEQ ID NO: 86 Amino acid sequence of complete lightchain of AM_(L)7 SEQ ID NO: 87 Nucleotide sequence of complete lightchain of AM_(L)7 SEQ ID NO: 88 Amino acid sequence encoding CDR1, CDR 2,CDR 3, SEQ ID NO: respectively, of heavy chain variable region AM_(H)189, 90, 91 Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)2 92, 93, 94 Aminoacid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, ofheavy chain variable region AM_(H)3 95, 96, 97 Amino acid sequenceencoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, of heavy chainvariable region AM_(H)4 98, 99, 100 Amino acid sequence encoding CDR1,CDR 2, CDR 3, SEQ ID NO: respectively, of heavy chain variable regionAM_(H)5 101, 102, 103 Amino acid sequence encoding CDR1, CDR 2, CDR 3,SEQ ID NO: respectively, of heavy chain variable region AM_(H)6 104,105, 106 Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)7 107, 108, 109 Aminoacid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, ofheavy chain variable region AM_(H)8 110, 111, 112 Amino acid sequenceencoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, ofheavy chainvariable region AM_(H)9 113, 114, 115 Amino acid sequence encoding CDR1,CDR 2, CDR 3, SEQ ID NO: respectively, of heavy chain variable regionAM_(H)10 116, 117, 118 Amino acid sequence encoding CDR1, CDR 2, CDR 3,SEQ ID NO: respectively, of heavy chain variable region AM_(H)11 119,120, 121 Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)12 122, 123, 124Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)13 125, 126, 127Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)14 128, 129, 130Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)15 131, 132, 133Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)16 134, 135, 136Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)17 137, 138, 139Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)1 140, 141, 142 Aminoacid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, oflight chain variable region AM_(L)2 143, 144, 145 Amino acid sequenceencoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, of light chainvariable region AM_(L)3 146, 147, 148 Amino acid sequence encoding CDR1,CDR 2, CDR 3, SEQ ID NO: respectively, of light chain variable regionAM_(L)4 149, 150, 151 Amino acid sequence encoding CDR1, CDR 2, CDR 3,SEQ ID NO: respectively, of light chain variable region AM_(L)5 152,153, 154 Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)6 155, 156, 157 Aminoacid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, oflight chain variable region AM_(L)7 158, 159, 160 Amino acid sequenceencoding CDR1, CDR 2, CDR 3, SEQ ID NO: respectively, of light chainvariable region AM_(L)8 161, 162, 163 Amino acid sequence encoding CDR1,CDR 2, CDR 3, SEQ ID NO: respectively, of light chain variable regionAM_(L)9 164, 165, 166 Amino acid sequence encoding CDR1, CDR 2, CDR 3,SEQ ID NO: respectively, of light chain variable region AM_(L)10 167,168, 169 Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)11 170, 171, 172Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)12 173, 174, 175Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)13 176, 177, 178Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)14 179, 180, 181Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)15 182, 183, 184Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)16 185, 186, 187Amino acid sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)17 188, 189, 190Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)1 191, 192, 193Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)2 194, 195, 196Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)3 197, 198, 199Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)4 200, 201, 202Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)5 203, 204, 205Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)6 206, 207, 208Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)7 209, 210, 211Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)8 212, 213, 214Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)9 215, 216, 217Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)10 218, 219, 220Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)11 221, 222, 223Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)12 224, 225, 226Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)13 227, 228, 229Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)14 230, 231, 232Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)15 233, 234, 235Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)16 236, 237, 238Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of heavy chain variable region AM_(H)17 239, 240, 241Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)1 242, 243, 244Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)2 245, 246, 247Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)3 248, 249, 250Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)4 251, 252, 253Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)5 254, 255, 256Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)6 257, 258, 259Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)7 260, 261, 262Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)8 263, 264, 265Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)9 266, 267, 268Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)10 269, 270, 271Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)11 272, 273, 274Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)12 275, 276, 277Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)13 278, 279, 280Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)14 281, 282, 283Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)15 284, 285, 286Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)16 287, 288, 289Nucleotide sequence encoding CDR1, CDR 2, CDR 3, SEQ ID NO:respectively, of light chain variable region AM_(L)17 290, 291, 292

2. Scaffolds

As noted herein, the antigen binding proteins can comprise a scaffoldstructure into which the CDR(s) are grafted. The scaffold structure maybe based on antibodies (including, but not limited to, monoclonalantibodies, human antibodies, murine antibodies, chimeric antibodies,humanized antibodies, bispecific antibodies), antibody fragments (suchas minibodies, domain antibodies, F(ab) fragments, F(ab′) fragments,F(ab)₂ fragments, F(ab′)₂ fragments, Fv fragments, scFv fragments, Fdfragments), synthetic antibodies (sometimes referred to herein as“antibody mimetics”), antibody fusions (sometimes referred to as“antibody conjugates”), including Fc fusions. Some embodiments includethe use of human scaffold components. The invention as such at leastencompasses any of the below described scaffolds comprising one orseveral of the CDRs as identified in SEQ ID NOs:89-190, preferably ofSEQ ID NOs:89-189, that can bind to and/or inhibit the biologicalactivity of IL-18 receptor. In some embodiments, the scaffold comprisesone or several heavy chain variable regions as identified in SEQ IDNOs:1-17, and or one or several light chain variable regions asidentified in any of SEQ ID NOs:18-34. In some embodiments, the scaffoldcomprises an IgG chain as identified in any of SEQ ID NOs:77-88.

In one embodiment, the scaffold into which one or several CDRs aregrafted is an antibody. As used herein, the term “antibody” refers to amultimeric protein having a traditional antibody structure, comprisingat least two, more typically four polypeptide chains. An antibody bindsspecifically to an antigen and may be able to inhibit or modulate thebiological activity of the antigen. In certain embodiments, antibodiesare produced by recombinant DNA techniques. In additional embodiments,antibodies are produced by enzymatic or chemical cleavage of naturallyoccurring antibodies.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). The amino-terminalportion of each chain includes a variable region of about 100 to 110 ormore amino acids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2.

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about twelve (12) or more amino acids, withthe heavy chain also including a “D” region of about ten (10) more aminoacids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch.7, 2nd ed. Raven Press, N.Y. The variable regions of each light/heavychain pair form the antibody binding site.

Some naturally occurring antibodies, for example found in camels andllamas, are dimers consisting of two heavy chain and include no lightchains. Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter etal., 2001, J. Biol. Chem. 276:26285-26290. Crystallographic studies of acamel antibody have revealed that the CDR3 regions form a surface thatinteracts with the antigen and thus is critical for antigen binding likein the more typical tetrameric antibodies.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three hypervariable regions, also called complementaritydetermining regions or CDRs. The CDRs are the hypervariable regions ofan antibody (or antigen binding protein, as outlined herein), which areresponsible for antigen recognition and binding. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest. Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia etal., 1989, Nature 342:878-883.

CDRs constitute the major surface contact points for antigen binding.See, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Further,CDR3 of the light chain and, especially, CDR3 of the heavy chain mayconstitute the most important determinants in antigen binding within thelight and heavy chain variable regions. See, e.g., Chothia and Lesk,1987, supra; Desiderio et al., 2001, J. Mol. Biol. 310:603-615; Xu andDavis, 2000, Immunity 13:37-45; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290; and Muyldermans, 2001, J. Biotechnol. 74:277-302. Insome antibodies, the heavy chain CDR3 appears to constitute the majorarea of contact between the antigen and the antibody. Desmyter et al.,2001, supra. In vitro selection schemes in which CDR3 alone is variedcan be used to vary the binding properties of an antibody. Muyldermans,2001, supra; Desiderio et al., 2001, supra.

Naturally occurring antibody chains typically include a signal sequence,which directs the antibody chain into the cellular pathway for proteinsecretion and which is not present in the mature antibody. Apolynucleotide encoding an antibody chain may encode a naturallyoccurring signal sequence or a heterologous signal sequence as describedbelow.

In one embodiment, the antigen binding protein is a monoclonal antibody,with from one (1) to six (6) of the depicted CDRs of any of SEQ IDNOs:89-190, as outlined herein. The antibodies of the invention may beof any type including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD,IgA, or IgE antibody. In specific embodiment, the antigen bindingprotein is an IgG type antibody. In an even more specific embodiment,the antigen binding protein is an IgG2 type antibody.

In some embodiments, for example when the antigen binding protein is anantibody with complete heavy and light chains, the CDRs are all from thesame species, e.g., human. In some embodiments, however, the scaffoldcomponents can be a mixture from different species. As such, if theantigen binding protein is an antibody, such antibody may be a chimericantibody and/or a humanized antibody. In general, both “chimericantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human.

For example in embodiments wherein the antigen binding protein containsless than six CDRs from the sequences outlined above, additional CDRsmay be either from other species (e.g., murine CDRs), or may bedifferent human CDRs than those depicted in the sequences. For example,human CDRH3 and CDRL3 regions from the appropriate sequences identifiedherein may be used, with CDRH1, CDRH2, CDRL1 and CDRL2 being optionallyselected from alternate species, or different human antibody sequences,or combinations thereof. For example, the CDRs of the invention canreplace the CDR regions of commercially relevant chimeric or humanizedantibodies.

Specific embodiments of the invention utilize scaffold components of theantigen binding proteins that are human components.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. Humanized antibodies canalso be generated using mice with a genetically engineered immunesystem. Roque et al., 2004, Biotechnol. Prog. 20:639-654. In the presentinvention, the identified CDRs are human, and thus both humanized andchimeric antibodies in this context include some non-human CDRs; forexample, humanized antibodies may be generated that comprise the CDRH3and CDRL3 regions, with one or more of the other CDR regions being of adifferent special origin.

In one embodiment, the IL-18 antigen binding protein is a multispecificantibody, and notably a bispecific antibody, also sometimes referred toas “diabodies”. These are antibodies that bind to two (or more)different antigens. Diabodies can be manufactured in a variety of waysknown in the art (Holliger and Winter, 1993, Current Opinion Biotechnol.4:446-449), e.g., prepared chemically or from hybrid hybridomas.

In one embodiment, the IL-18 antigen binding protein is a fully humanantibody, i.e., an antibody fully composed of human components. In thisembodiment, as outlined above, specific structures comprise completeheavy and light chains depicted comprising the CDR regions depicted inFIGS. 2A-2E, 3A and 3B. Additional embodiments utilize one or more ofthe CDRs of the invention, with the other CDRs, framework regions, J andD regions, constant regions, etc., coming from other human antibodies.For example, the CDRs of the invention can replace the CDRs of anynumber of human antibodies, particularly commercially relevantantibodies.

In one embodiment, the IL-18 antigen binding protein is an antibodyfragment, that is a fragment of any of the antibodies outlined hereinthat retain binding specificity to an α- or β-IL-18 receptor.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fab′fragment consisting of VL, VH, CL and CH1 domains plus the heavy chainhinge region; (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546)which consists of a single variable, (v) isolated CDR regions, (vi)F(ab′)₂ fragments, a bivalent fragment comprising two linked Fab′fragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988,Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965)and (ix) “diabodies” or “triabodies”, multivalent or multispecificfragments constructed by gene fusion (Tomlinson et. al., 2000, MethodsEnzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified.For example, the molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al., 1996,Nature Biotech. 14:1239-1245). Again, as outlined herein, the non-CDRcomponents of these fragments are preferably human sequences.

In one embodiment, the IL-18 antigen binding protein is a minibody.Minibodies are minimized antibody-like proteins comprising a scFv joinedto a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

In one embodiment, the IL-18 antigen binding protein is a domainantibody; see for example U.S. Pat. No. 6,248,516. Domain antibodies(dAbs) are functional binding domains of antibodies, corresponding tothe variable regions of either the heavy (VH) or light (VL) chains ofhuman antibodies dABs have a molecular weight of approximately 13 kDa,or less than one-tenth the size of a full antibody. dABs are wellexpressed in a variety of hosts including bacterial, yeast, andmammalian cell systems. In addition, dAbs are highly stable and retainactivity even after being subjected to harsh conditions, such asfreeze-drying or heat denaturation. See, for example, U.S. Pat. Nos.6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941;European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821,WO04/003019 and WO03/002609.

In one embodiment, the IL-18 antigen binding protein is an antibodyfusion protein or an antibody fragment fusion, such as an Fc fusion(sometimes collectively referred to herein as an “antibody conjugate”).The conjugate partner can be proteinaceous or non-proteinaceous; thelatter generally being generated using functional groups on the antigenbinding protein (see the discussion on covalent modifications of theantigen binding proteins) and on the conjugate partner. For examplelinkers are known in the art; for example, homo- or hetero-bifunctionallinkers as are well known (see, 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference).

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antigen binding proteins, or binding of a radionuclideto a chelating agent that has been covalently attached to the antigenbinding protein. Additional embodiments utilize calicheamicin,auristatins, geldanamycin and maytansine.

In one embodiment, the IL-18 antigen binding protein is an antibodyanalog, sometimes referred to as “synthetic antibodies.” For example, avariety of recent work utilizes either alternative protein scaffolds orartificial scaffolds with grafted CDRs. Such scaffolds include, but arenot limited to, mutations introduced to stabilize the three-dimensionalstructure of the binding protein as well as wholly synthetic scaffoldsconsisting for example of biocompatible polymers. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold. Alternate scaffoldsthat may be used to produce an IL-18 antigen binding protein arereviewed in Hey et al., 2005, Trends Biotechnol. 23:514-22 and Binz etal., Nature Biotechnology 23:1257-68 (both incorporated herein byreference in their entirety).

3. CDR Variants

Also included within the invention are variants of the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences depicted in SEQ IDNOs:89-190. Thus variant CDRs are included within the definition of CDRas used herein. These variants fall into one or more of three classes:substitutional, insertional or deletional variants, with the formerbeing specific.

As it is known in the art, a number of different programs can be used toidentify the degree of sequence identity or similarity a protein ornucleic acid has to a known sequence.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 80% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, and almost 100%.

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the nucleic acid sequence of the binding proteins identifiedherein is defined as the percentage of nucleotide residues in acandidate sequence that are identical with the nucleotide residues inthe coding sequence of the antigen binding protein. A specific methodutilizes the BLASTN module of WU-BLAST-2 set to the default parameters,with overlap span and overlap fraction set to 1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 60%, and moretypically with preferably increasing homologies or identities of atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, and almost 100%.

Homology between nucleotide sequences is often defined by their abilityto hybridize to each other. The term “selectively hybridize” referred toherein means to detectably and specifically bind. Polynucleotides,oligonucleotides and fragments thereof in accordance with the inventionselectively hybridize to nucleic acid strands under hybridization andwash conditions that minimize appreciable amounts of detectable bindingto nonspecific nucleic acids. High stringency conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein.

High stringency conditions are known in the art; see, for exampleSambrook et al., 2001, supra, and Short Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, both ofwhich are hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques In Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993).

Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium Ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see, Sambrook et al., 2001, supra; Ausubel et al.,1992, supra, and Tijssen, 1993, supra.

The variants according to the invention are ordinarily prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the antigenbinding protein, using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the recombinant DNA in cell culture as outlinedherein. However, antigen binding protein fragments comprising variantCDRs having up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, e.g., binding to IL-18 receptor and inhibiting signaling,although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 90, 91, 92, 93, 94,95, 96, 97, 98% or 99% of the specificity and/or activity of the parentCDR. For example, the variants typically will bind to the same IL-18receptor epitope outlined below, with a similar inhibition of IL-18receptor signaling.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding protein activities, such as IL-18 receptorbinding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. When small alterations in the characteristics of the CDRof the antigen binding protein are desired, substitutions are generallymade in accordance with the following chart depicted as TABLE 2.

TABLE 2 Original Residues Exemplary Substitution Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTABLE 2. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the antigen binding protein proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the antigen binding protein is altered. For example,glycosylation sites may be altered or removed as discussed herein.

4. VH And VL Variants

As outlined above, in some embodiments the invention provides antigenbinding proteins comprising, or consisting of, a heavy chain variableregion of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, or 17 and/or a light chain variable region of SEQ ID NO:18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, respectively,or fragments thereof as defined above. Thus, in those embodiments, theantigen binding protein comprises not only at least one CDR or variantthereof depicted in SEQ ID NOs:1-34, but also at least part of adepicted framework sequence. In addition, the invention encompassesvariants of such heavy chain variable sequences or light chain variablesequences.

A “variant VH” or “variant heavy chain variable region,” and a “variantVL” or “variant light chain variable region” generally shares an aminoacid homology, similarity, or identity of at least 80% with thosedepicted herein, and more typically with preferably increasinghomologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% and almost 100%. The nucleic acid sequence homology,similarity, or identity between the nucleotide sequences encodingindividual variant VHs and VLs and the nucleic acid sequences depictedherein are at least 60% with those depicted herein, and more typicallywith preferably increasing homologies or identities of at least 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% andalmost 100%. In addition, a “variant VH” or “variant heavy chainvariable region,” and a “variant VL” or “variant light chain variableregion” typically shares the biological function, including, but notlimited to, at least 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% of thespecificity and/or activity of the parent CDR. For example, the variantstypically will bind to the same IL-18 receptor epitopes outlined below,with a similar inhibition of IL-18 receptor signaling.

Methods of generating variants, as well as methods of determiningsequence homology, similarity, and identity, are outlined supra, seeSection V.B.1.

In some embodiments, constant region variants may also be included.Preferred constant region variants include those that alter a biologicalfunction of the antibody containing the variation. For example, theantibody may contain a variation that alters the antibody's ability toactivate complement or induce antibody-dependent cellular cytotoxicity(ADCC). Such variants may include those that result in an alteration ofthe glycosylation of the antibody.

C. IL-18 Receptor and IL-18 Receptor Epitopes

By “IL-18 receptor” or “IL-18R” herein is meant the cell surfacereceptor that binds to a ligand, including, but not limited to, IL-18and as a result initiates a signal transduction pathway within the cell.The IL-18 receptor complex is made up of an IL-18 binding chain termed“α-IL-18 receptor” (α-IL-18R) or “IL-18Rα chain,” and a signaling chain,termed β-IL-18 receptor “β-IL-18 receptor” (“β-IL-18R”) or “IL-18Rβchain.” As used herein, the term “IL-18 receptor” collectively refers toboth the α- and the β-IL-18 receptor.

The antigen binding proteins disclosed herein bind to the IL-18Rα chain,the human amino acid sequence of which is depicted in SEQ ID NO:69 (itsnucleic acid sequence is depicted in SEQ ID NO:70), or the IL-18Rβchain, the human amino acid sequence of which is depicted in SEQ IDNO:71 (its nucleic acid sequence is depicted in SEQ ID NO:72). In aspecific embodiment, the IL-18 receptor is human, although in somecases, other species may be used. In addition, as described below, IL-18receptor proteins may also include fragments.

As is described below, binding of antigen binding proteins to specificepitopes is specific.

By “epitope”, “antigenic determinant”, and grammatical equivalentsherein are meant a region of an antigen, e.g., IL-18 receptor, which canbe specifically bound by an antigen binding protein. As the skilledartisan will appreciate, an epitope can be linear or conformational.“Linear epitope” refers to an epitope comprising a sequence of at leastabout five (5) and not more than about twenty (20) amino acids connectedin a linear fashion, which amino acids, by themselves or as part of alarger sequence, bind to an antigen binding protein of the invention.“Conformational epitope” refers to an epitope whose three dimensional,secondary and/or tertiary structure can be a substantial aspect ofantibody binding. Generally but not uniformly, amino acids that comprisea conformational epitope do not comprise a linear sequence of aprotein's primary structure. Thus, a conformational epitope may beshared by proteins having non-homologous linear amino acid sequences.Without being bound by theory, a conformational epitope can be sharedbecause the tertiary structure recognized by an antibody can be sharedbetween two or more amino acid sequences. In one embodiment, suitableIL-18 receptor epitopes include any which are recognized by the antigenbinding proteins of the present invention.

The invention provides antigen binding proteins recognizing and bindingto a conformational epitope in the third Ig domain of human IL-18Rα, inparticular, the region defined by amino acid residues 243-271, made upby amino acid residues 250-253 (i.e., residues MFGE) and amino acidresidues 267-271 (i.e., residues MRIMT) of SEQ ID NO:69. The amino acidstructure of this epitope is depicted in FIG. 5. Antigen bindingproteins that bind to this epitope are particularly effective atblocking the interaction of IL-18 with the IL-18R. Methods ofdetermining the binding epitope of an antigen binding protein are wellknown in the art and one such method is described in Example 4 herein.

Example 4 demonstrates that certain human IL-18R antigen bindingproteins had significantly reduced ability to bind the human IL-18Rαwhen residues within the epitope defined by amino acids 243-271, e.g.,250-253 or 267-271, were changed to the corresponding mouse residues.Thus, provided herein are antigen binding proteins that bind humanIL-18R but such binding is reduced when residues 250-253 of the humanIL-18Rα chain are substituted with the corresponding mouse amino acids.Also provided herein are antigen binding proteins that bind human IL-18Rbut such binding is reduced when residues 267-271 of the human IL-18Rαchain are substituted with the corresponding mouse amino acids.

D. Covalent Modifications of Antigen Binding Protein

Covalent modifications of antigen binding proteins are included withinthe scope of this invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antigen binding protein are introduced into themolecule by reacting specific amino acid residues of the antigen bindingprotein with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

1. Glycosylation

Another type of covalent modification of the antigen binding proteinincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

2. PEGylation

Another type of covalent modification of the antigen binding proteincomprises linking the antigen binding protein to variousnonproteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, asis known in the art, amino acid substitutions may be made in variouspositions within the antigen binding protein to facilitate the additionof polymers such as PEG.

3. Labels and Effector Groups

In some embodiments, the covalent modification of the antigen bindingproteins of the invention comprises the addition of one or more labels.

The term “labeling group” means any detectable label. Examples ofsuitable labelling groups include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labelling group is coupled to theantigen binding protein via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and may be used in performing the present invention.

The term “effector group” means any group coupled to an antigen bindingprotein that acts as a cytotoxic agent. Examples for suitable effectorgroups are radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I). Other suitable groups include toxins,therapeutic groups, or chemotherapeutic groups. Examples of suitablegroups include calicheamicin, auristatins, geldanamycin and maytansine.In some embodiments, the effector group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabelling proteins are known in the art and may be used in performingthe present invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

E. Polynucleotides Encoding IL-18 Receptor Antigen Binding Proteins

In certain aspects, the invention provides nucleic acid moleculesencoding the IgGs, variable regions and CDRs of SEQ ID NOs:1-34, 73, 75,77, 79, 81, 83, 85, 87, 89-190. In one embodiment, the nucleic acidshave the nucleotide sequence of any of SEQ ID NOs:35-68, 74, 76, 78, 80,82, 84, 86, 88, and 191-292.

As described herein, a variable region or CDR nucleic acid encodes avariable region or CDR protein, respectively. By “nucleic acid” hereinis meant any nucleic acid, including both DNA and RNA. Nucleic acids ofthe present invention are typically polynucleic acids; that is, polymersof individual nucleotides that are covalently joined by 3′, 5′phosphodiester bonds.

Depending on its use, the nucleic acid may be double stranded, singlestranded, or contain portions of both double stranded or single strandedsequence. As will be appreciated by those in the art, the depiction of asingle strand (“Watson”) also defines the sequence of the other strand(“Crick”); thus the nucleic acid sequences depicted in SEQ ID NOs:35-68also include the complement of these sequences. By the term “recombinantnucleic acid” herein is meant nucleic acid, originally formed in vitro,in general, by the manipulation of nucleic acid by endonucleases, in aform not normally found in nature. Thus an isolated antigen bindingprotein nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.,using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antigen binding protein) of the present invention.Thus, having identified a particular amino acid sequence, such as SEQ IDNOs:1-34, those skilled in the art could make any number of differentnucleic acids, by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the encodedprotein.

F. Methods of Producing Antigen Binding Proteins

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the IL-18receptor antigen binding protein coding sequence; the oligonucleotidesequence encodes polyHis (such as hexaHis), or another “tag” such asFLAG, HA (hemaglutinin influenza virus), or myc, for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as a meansfor affinity purification or detection of the IL-18 receptor antigenbinding protein from the host cell. Affinity purification can beaccomplished, for example, by column chromatography using antibodiesagainst the tag as an affinity matrix. Optionally, the tag cansubsequently be removed from the purified IL-18 receptor antigen bindingprotein by various means such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein antibody that binds to IL-18 receptorpolypeptide. As a result, increased quantities of a polypeptide such asan IL-18 receptor antigen binding protein are synthesized from theamplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the IL-18 receptor antigen binding protein.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an IL-18 receptor antigen bindingprotein of the invention by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the desired promoter sequenceinto the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin genecontrol region that is active in pancreatic beta cells (Hanahan, 1985,Nature 315:115-122); the immunoglobulin gene control region that isactive in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising anIL-18 receptor antigen binding protein of the invention by highereukaryotes. Enhancers are cis-acting elements of DNA, usually about10-300 bp in length, that act on the promoter to increase transcription.Enhancers are relatively orientation and position independent, havingbeen found at positions both 5′ and 3′ to the transcription unit.Several enhancer sequences available from mammalian genes are known(e.g., globin, elastase, albumin, alpha-feto-protein and insulin).Typically, however, an enhancer from a virus is used. The SV40 enhancer,the cytomegalovirus early promoter enhancer, the polyoma enhancer, andadenovirus enhancers known in the art are exemplary enhancing elementsfor the activation of eukaryotic promoters. While an enhancer may bepositioned in the vector either 5′ or 3′ to a coding sequence, it istypically located at a site 5′ from the promoter. A sequence encoding anappropriate native or heterologous signal sequence (leader sequence orsignal peptide) can be incorporated into an expression vector, topromote extracellular secretion of the antibody. The choice of signalpeptide or leader depends on the type of host cells in which theantibody is to be produced, and a heterologous signal sequence canreplace the native signal sequence. Examples of signal peptides that arefunctional in mammalian host cells include the following: the signalsequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195;the signal sequence for interleukin-2 receptor described in Cosman etal., 1984, Nature 312:768; the interleukin-4 receptor signal peptidedescribed in EP Patent No. 0367 566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; the type IIinterleukin-1 receptor signal peptide described in EP Patent No. 0 460846.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an IL-18 receptor antigen binding sequence has been insertedinto the proper site of the vector, the completed vector may be insertedinto a suitable host cell for amplification and/or polypeptideexpression. The transformation of an expression vector for an IL-18receptor antigen binding protein into a selected host cell may beaccomplished by well known methods including transfection, infection,calcium phosphate co-precipitation, electroporation, microinjection,lipofection, DEAE-dextran mediated transfection, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anIL-18 receptor antigen binding protein that can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). The selection of an appropriate host cell will depend uponvarious factors, such as desired expression levels, polypeptidemodifications that are desirable or necessary for activity (such asglycosylation or phosphorylation) and ease of folding into abiologically active molecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with IL-18 receptorbinding properties. In another embodiment, a cell line from the B celllineage that does not make its own antibody but has a capacity to makeand secrete a heterologous antibody can be selected.

G. Use of IL-18 Receptor Antigen Binding Proteins for Diagnostic andTherapeutic Purposes

Antigen binding proteins of the invention are useful for detecting IL-18receptor in biological samples and identification of cells or tissuesthat produce IL-18 receptor protein. Antigen binding proteins of theinvention that specifically bind to IL-18 receptor may be used intreatment of IL-18 receptor mediated diseases in a patient in needthereof. For one, the IL-18 receptor antigen binding proteins of theinvention can be used in diagnostic assays, e.g., binding assays todetect and/or quantify IL-18 receptor expressed in a tissue or cell. Inaddition, the IL-18 receptor antigen binding protein of the inventioncan be used to inhibit IL-18 receptor from forming a complex with itsligand, e.g., IL-18, thereby modulating the biological activity of IL-18receptor in a cell or tissue. Antigen binding proteins that bind toIL-18 receptor thus may modulate and/or block interaction with otherbinding compounds and as such may have therapeutic use in amelioratingIL-18 receptor mediated diseases. In specific embodiments, IL-18receptor antigen binding proteins may block IL-18 binding to itsreceptor, which may result in disruption of the IL-18 receptor inducedsignal transduction cascade.

1. Indications

Increased levels of IL-18 and/or involvement of IL-18 mediated signalsin disease pathogenesis have been demonstrated in a variety ofconditions and diseases. The antigen binding proteins of the presentinvention thus serve to regulate or suppress an immune response and haveefficacy in the treatment and prevention of various diseases caused byan excessive immune response (see, WO2004/002519; WO2005/063290;WO2004/034988; Mallat et al., 2002, Circ. Res. 91:441-448). Accordingly,the IL-18 receptor antigen binding proteins of the present invention canbe used for the diagnosis, prevention or treatment of diseases orconditions associated with the IL-18.

A disease or condition associated with IL-18 means any disease orcondition whose onset in a patient is caused by or prevented by theinteraction of IL-18 with the IL-18 receptor. The severity of thedisease or condition can also be increased or decreased by theinteraction of IL-18 with the IL-18 receptor. For example, IL-18 isassociated with autoimmune diseases (WO2004/002519; WO2005/063290;WO2004/034988; Mallat et al., 2002, Circ. Res. 91:441-448), hepaticdisease (Finitto et al., 2004, Liver 53:392-400; Tsutsui et al., 2000,Immunological Reviews 174:192-209; Ludwiczek et al., 2002, J. ClinicalImmunology 22:331-337), pancreatic disease and cardiovascular diseases(Gerdes et al, 2002, J. Exp. Med. 195:245-257; WO03/080104; WO02/060479;WO01/85201; Raeburn et al., 2002, Am. J. Physiol. Heart Circ. Physiol.283:H650-H657).

Examples of autoimmune diseases that are associated with IL-18 includepsoriasis, inflammatory arthritis such as rheumatoid arthritis(WO2005/063290; Cannetti et al., 2003, J. Immunol. 171:1009-1015;Charles et al., 1999, J. Immunol. 163: 1521-1528; Cunnane et al., 2000,Online J. Rheumatol. 27:58-63; Yoshimoto, 1998, J. Immunol. 161:3400-3407), lupus (WO2005/063290), Type I diabetes, Type II diabetes,Crohn's disease (Niederau, 1997, Online NLM), inflammatory bowel disease(WO2004/002519), multiple sclerosis, autoimmune hepatitis (Tsutsui etal., 2000, supra), primary biliary cirrhosis (PBC), acquired immunedeficiency syndrome (AIDS), atopic dermatitis (Konishi et al., 2002,Proc. Natl. Acad. Sci. U.S.A. 99:11340-11345), myasthenia gravis, andsarcoidosis.

In rheumatoid arthritis, elevated levels of mature IL-18 have beendemonstrated in patient sera and synovial fluid. In some studies, IL-18levels were shown to correlate with disease activity and response todisease modifying treatment. Extremely elevated serum levels of IL-18have consistently been measured in systemic Juvenile IdiopathicArthritis and the closely related Adult-Onset Still's Disease.WO2005/063290; Cannetti et al., 2003, J. Immunol. 171:1009-1015; Charleset al., 1999, J. Immunol. 163: 1521-1528; Cunnane et al., 2000, OnlineJ. Rheumatol. 27:58-63; Yoshimoto, 1998, J. Immunol. 161: 3400-3407.

Other forms of arthritis that are associated with IL-18 include forexample ankylosing spondylitis, back pain, carpal deposition syndrome,Ehlers-Danlos-Syndrome, gout, juvenile arthritis, lupus erythematosus,myositis, osteogenesis imperfecta, osteoporosis, polyartheritis;polymyositis, psoriatic arthritis, Reiter's syndrome, scleroderma,arthritis with bowel disease, Behcets's disease, children's arthritis,degenerative joint disease, fibromyalgia, infectious arthritis, Lymedisease, Marfan syndrome, osteoarthritis, osteonecrosis, Pagets Disease,Polymyalgia rheumatica, pseudogout, reflex sympathetic dystrophy,rheumatoid arthritis, rheumatism, Sjogren's syndrome, familialadenomatous polyposis and the like. Dai et al., 2004, Arthritis Rheum.50:432-443; Kawashima et al., 2004, Online Arthritis Res. Ther.6:R39-R45; Myers et al., 2004, Rheumatology 43:272-276; Wei et al.,2001, American Association Of Immunologists, pp. 517-521.

Elevated levels of IL-18 have also been found in patients with Crohn'sdisease when compared with patients with ulcerative colitis ornon-inflammatory intestinal conditions. Both intestinal epithelial cellsand lamina propria mononuclear cells have been identified as the sourceof increased IL-18 production in situ. Crohn's disease lesions have beenshown to be infiltrated with IL-18R expressing cells. Niederau, 1997,Online NLM.

IL-18 has also been implicated as being associated with ulcerativecolitis and Coeliac Disease.

Central Nervous System (CNS) lesions, cerebrospinal fluid, and sera frompatients with Multiple Sclerosis have been shown to contain increasedlevels of IL-18 message or protein. Within lesions, microglia andmacrophages are thought to be the source of IL-18. IL-18 cannot bedetected in control tissue biopsies from individuals withnon-inflammatory CNS diseases. Particularly high levels of IL-18 hasbeen found in the patient subset with relapsing-remitting disease; andIL-18 levels have been found to increase during relapses compared toperiods of remission. Huang et al., 2004, Mult. Scler. 10:482-7; Karniet al., 2002, J. Neuroimmunol. 125:134-40; Losy et al., 2001, ActaNeurol. Scand. 104:171-3; Nicoletti et al., 2001, Neurology 57:342-4;Fassbender et al., 1999, Neurology 53:1104-6.

In patients with psoriasis, serum levels of IL-18 were reported to beincreased, correlating with the extent of skin lesions and PASI score.Overexpression of both IL-18 and IL-18R mRNA has been demonstrated inlesional skin compared with non-lesional or normal skin controls.Documented overexpression of IFN-γ and TNF-α in psoriatic skin isconsistent with biological activities exerted by IL-18. Arican et al.,2005, Mediators Inflamm. 2005:273-9; Piskin et al., 2004, Exp. Dermatol.13:764-72; Companjen et al., 2004 Eur. Cytokine Netw. 15:210-6; Pietrzaket al., 2003, Acta Derm. Venereol. 83:262-5.

Various other autoimmune diseases have been associated with increasedlevels of IL-18 either in diseased tissue or in the serum. These includeSystemic Lupus Erythematosus, atopic dermatitis, myasthenia gravis, typeI diabetes, and sarcoidosis. IL-18 may also be involved in asthma,Alzheimer's Disease, allergic rhinitis, Idiopathic ThrombocytopenicPurpura (ITP), transplantation and GvHD.

IL-18 has also been implicated in a liver or hepatic diseases and inconditions associated with liver damage or injury. Liver damage orinjury may have diverse causes. It may be due to viral or bacterialinfections, alcohol abuse, immunological disorders, or cancer, forexample. Liver injury also includes damages of the bile ducts, anddamage to the liver in conditions such as alcoholic hepatitis, livercirrhosis, viral hepatitis, primary biliary cirrhosis, andalcohol-related hepatic necro-inflammation. Finitto et al., 2004, Liver53:392-400; Tsutsui et al., 2000, Immunological Reviews 174:192-209;Ludwiczek et al., 2002, J. Clinical Immunology 22:331-337.

Hepatic diseases that are associated with IL-18 include hepatitis C andhepatitis B. IL-18 has been implicated in the pathogenesis of bothautoimmune and infectious hepatitis. It is thought to contribute tohepatocyte death via upregulation of proapoptotic molecules, includingFasL. It has been suggested that the beneficial effect ofinterferon-alpha in hepatitis C may be mediated through reduced levelsof IL-18. In contrast, IL-18 administration had a beneficial effect in atransgenic model of hepatitis B, improving viral clearance throughincreased NK and CTL activity. Finitto et al., 2004, Liver 53:392-400;Tsutsui et al., 2000, Immunological Reviews 174:192-209; Ludwiczek etal., 2002, J. Clinical Immunology 22:331-337.

Apart from Hepatitis B and C virus, at least four other viruses causingvirus-associated hepatitis have been discovered so far, called HepatitisA, D, E and G-Virus.

IL-18 is also associated with cardiovascular disease, includingatheromatous plaque rupture, post-ischemic heart failure, reperfusioninjury, atherosclerosis, chronic heart failure, cardiovascularcomplications of rheumatoid arthritis and other cardiovasculardisorders. IL-18 is thought to markedly depress cardiac output in thesetting of sepsis or endotoxin shock. IL-18 is an important link betweeninflammatory processes and atherogenesis, which is particularly relevantgiven the accumulating evidence for large excess mortality fromcardiovascular causes in patients with chronic inflammatory conditions,including RA and lupus. IL-18 levels have been shown to be a strongindependent predictor of death from cardiac events (with betterpredictive power than CRP levels). Gerdes et al, 2002, J. Exp. Med.195:245-257; WO03/080104; WO02/060479; WO01/85201; Raeburn et al., 2002,Am. J. Physiol. Heart Circ. Physiol. 283:H650-H657.

IL-18 may also be associated with pulmonary diseases such as, forexample, Chronic Obstructed Pulmonary Disease (COPD), chronic severeasthma and Acute Respiratory Distress Syndrome (ARDS).

2. Diagnostic Methods

The antigen binding proteins of the invention can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or conditionsassociated with IL-18 or the IL-18 receptor. The invention provides forthe detection of the presence of the IL-18 receptor in a sample usingclassical immunohistological methods known to those of skill in the art(e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, vol 15(Eds R. H. Burdon and P. H. van Knippenberg, Elsevier, Amsterdam); Zola,1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRCPress, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101:976-985;Jalkanen et al., 1987, J. Cell Biol. 105:3087-3096). The detection ofthe IL-18 receptor can be performed in vivo or in vitro.

Diagnostic applications provided herein include use of the antigenbinding proteins to detect expression of the IL-18 receptor and bindingof the ligands to the IL-18 receptor. Examples of methods useful in thedetection of the presence of the IL-18 receptor include immunoassays,such as the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically willbe labeled with a detectable labeling group. Suitable labeling groupsinclude, but are not limited to, the following: radioisotopes orradionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I),fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In someembodiments, the labelling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand may be used in performing the present invention.

One aspect of the invention provides for identifying a cell or cellsthat express the IL-18 receptor. In a specific embodiment, the antigenbinding protein is labeled with a labeling group and the binding of thelabeled antigen binding protein to the IL-18 receptor is detected. In afurther specific embodiment, the binding of the antigen binding proteinto the IL-18 receptor detected in vivo. In a further specificembodiment, the antigen binding protein-IL-18 receptor is isolated andmeasured using techniques known in the art. See, for example, Harlow andLane, 1988, Antibodies: A Laboratory Manual, New York: Cold SpringHarbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993,Current Protocols In Immunology New York: John Wiley & Sons.

Another aspect of the invention provides for detecting the presence of atest molecule that competes for binding to the IL-18 receptor with theantigen binding proteins of the invention. An example of one such assaywould involve detecting the amount of free antigen binding protein in asolution containing an amount of IL-18 receptor in the presence orabsence of the test molecule. An increase in the amount of free antigenbinding protein (i.e., the antigen binding protein not bound to theIL-18 receptor) would indicate that the test molecule is capable ofcompeting for IL-18 receptor binding with the antigen binding protein.In one embodiment, the antigen binding protein is labeled with alabeling group. Alternatively, the test molecule is labeled and theamount of free test molecule is monitored in the presence and absence ofan antigen binding protein.

3. Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

In some embodiments, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of one or a plurality ofthe antigen binding proteins of the invention together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In addition, the invention providesmethods of treating a patient by administering such pharmaceuticalcomposition. The term “patient” includes human and animal subjects.

Preferably, acceptable formulation materials are nontoxic to recipientsat the dosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of IL-18 receptor antigen binding proteins are provided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins of the invention. In certain embodiments, theprimary vehicle or carrier in a pharmaceutical composition may be eitheraqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. In specific embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,and may further include sorbitol or a suitable substitute therefor. Incertain embodiments of the invention, IL-18 receptor antigen bindingprotein compositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the IL-18 receptor antigen binding protein product may beformulated as a lyophilizate using appropriate excipients such assucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired IL-18 receptor antigen binding protein in a pharmaceuticallyacceptable vehicle. A particularly suitable vehicle for parenteralinjection is sterile distilled water in which the IL-18 receptor antigenbinding protein is formulated as a sterile, isotonic solution, properlypreserved. In certain embodiments, the preparation can involve theformulation of the desired molecule with an agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid or polyglycolic acid), beads or liposomes, that mayprovide controlled or sustained release of the product which can bedelivered via depot injection. In certain embodiments, hyaluronic acidmay also be used, having the effect of promoting sustained duration inthe circulation. In certain embodiments, implantable drug deliverydevices may be used to introduce the desired antigen binding protein.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, IL-18 receptor antigen bindingproteins are advantageously formulated as a dry, inhalable powder. Inspecific embodiments, IL-18 receptor antigen binding protein inhalationsolutions may also be formulated with a propellant for aerosol delivery.In certain embodiments, solutions may be nebulized. Pulmonaryadministration and formulation methods therefore are further describedin International Patent Application No. PCT/US94/001875, which isincorporated by reference and describes pulmonary delivery of chemicallymodified proteins. It is also contemplated that formulations can beadministered orally. IL-18 receptor antigen binding proteins that areadministered in this fashion can be formulated with or without carrierscustomarily used in the compounding of solid dosage forms such astablets and capsules. In certain embodiments, a capsule may be designedto release the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the IL-18 receptor antigen binding protein.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

A pharmaceutical composition of the invention is preferably provided tocomprise an effective quantity of one or a plurality of IL-18 receptorantigen binding proteins in a mixture with non-toxic excipients that aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions may be preparedin unit-dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving IL-18 receptor antigenbinding proteins in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, International PatentApplication No. PCT/US93/00829, which is incorporated by reference anddescribes controlled release of porous polymeric microparticles fordelivery of pharmaceutical compositions. Sustained-release preparationsmay include semipermeable polymer matrices in the form of shapedarticles, e.g., films, or microcapsules. Sustained release matrices mayinclude polyesters, hydrogels, polylactides (as disclosed in U.S. Pat.No. 3,773,919 and European Patent Application Publication No. EP 058481,each of which is incorporated by reference), copolymers of L-glutamicacid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers2:547-556), poly(2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J.Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., 1981, supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an IL-18 receptor antigenbinding protein-containing pharmaceutical composition to be employedwill depend, for example, upon the therapeutic context and objectives.One skilled in the art will appreciate that the appropriate dosagelevels for treatment will vary depending, in part, upon the moleculedelivered, the indication for which the IL-18 receptor antigen bindingprotein is being used, the route of administration, and the size (bodyweight, body surface or organ size) and/or condition (the age andgeneral health) of the patient. In certain embodiments, the clinicianmay titer the dosage and modify the route of administration to obtainthe optimal therapeutic effect. A typical dosage may range from about0.1 μg/kg to up to about 30 mg/kg or more, depending on the factorsmentioned above. In specific embodiments, the dosage may range from 0.1μg/kg up to about 30 mg/kg, optionally from 1 μg/kg up to about 30 mg/kgor from 10 μg/kg up to about 5 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular IL-18 receptor antigen binding protein in the formulationused. Typically, a clinician administers the composition until a dosageis reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of the desired molecule)over time, or as a continuous infusion via an implantation device orcatheter. Further refinement of the appropriate dosage is routinely madeby those of ordinary skill in the art and is within the ambit of tasksroutinely performed by them. Appropriate dosages may be ascertainedthrough use of appropriate dose-response data. In certain embodiments,the antigen binding proteins of the invention can be administered topatients throughout an extended time period. Chronic administration ofan antigen binding protein of the invention minimizes the adverse immuneor allergic response commonly associated with antigen binding proteinsthat are not fully human, for example an antibody raised against a humanantigen in a non-human animal, for example, a non-fully human antibodyor non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

It also may be desirable to use IL-18 receptor antigen binding proteinpharmaceutical compositions according to the invention ex vivo. In suchinstances, cells, tissues or organs that have been removed from thepatient are exposed to IL-18 receptor antigen binding proteinpharmaceutical compositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In particular, IL-18 receptor antigen binding proteins can be deliveredby implanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptide. In certain embodiments, such cells may be animal or humancells, and may be autologous, heterologous, or xenogeneic. In certainembodiments, the cells may be immortalized. In other embodiments, inorder to decrease the chance of an immunological response, the cells maybe encapsulated to avoid infiltration of surrounding tissues. In furtherembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

All references cited within the body of the instant specification arehereby expressly incorporated by reference in their entirety.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the invention.

VI. EXAMPLES A. Example 1 Production of IgG2 and IgG4 Versions ofAnti-IL-18 Receptor Antibodies Using pVE414N Transient ExpressionConstructs

The following example describes the generation of transient expressionconstructs used to produce IgG2 and IgG4 versions of various anti-IL18receptor binding proteins, and experimental approaches to test theirbinding characteristics and potency.

1. Generation of Constructs

Expression constructs for transient expression of IgG4 versions ofAM_(H)9/AM_(L)9, AM_(H)11/AM_(L)7, AM_(H)3/AM_(L)14, andAM_(H)6/AM_(L)12 were generated by subcloning the polynucleotidesequences of SEQ ID NOs:74, 76, 78, 80, 82, 84, 86 and 88 into atransient expression vector. IgG2 versions of the same variable regionswere generated by subcloning the polynucleotide portions encoding thevariable regions of those IgGs into a separate transient expressionvector.

2. Transient Roller Bottle Tranfections

Eight roller bottle transfections into CosPKB cell line for each of theantibodies were performed. The titers for the IgG2's were as follows:

IgG2 Titer AM_(H)9/AM_(L)9 33.5 AM_(H)11/AM_(L)7 35.1 AM_(H)3/AM_(L)1442.9 AM_(H)6/AM_(L)12 41.5

3. Assays for Potency and Cross Reactivity of Various Antibodies

KG-1 IFNγ Release Assay.

The various IgG constructs were tested to determine their inhibitoryactivity in an in vitro Interferon-γ (IFNγ) release assay. The IFNγrelease assay works on the principle that human myelomonocytic KG-1cells that express the endogenous IL-18R release IFNγ in response toIL-18.

Briefly, reagents such as affinity-purified scFv are pre-incubated withKG-1 cells in a 96-well tissue culture plate. IL-18 (+TNFα) is added tothe cells to induce IFNγ release. TNFα is added with the IL-18 toincrease the IFNγ response and therefore makes the assay more sensitiveby allowing a lower concentration of IL-18 to be used. This is at leastin part, likely due to TNFα induced upregulation of IL-18R surfaceexpression.

After a defined incubation period, the cell supernatants are harvestedand analyzed for IFNγ content using ELISA. Test compounds that inhibitIL-18R mediated signaling can be assessed in this assay by showing areduction in IFNγ release.

KG-1 cells were obtained from the European Collection of Cell Cultures(ECACC, 86111306). The cells were propagated in supplemented Iscove'sDulbecco modified medium (IMDM), and were maintained at 1−2×10⁶cells/ml. Recombinant human IL-18 was obtained from Peprotech (200-18)and the recombinant human TNFα was purchased from R&D Systems (210-TA).The amount of IFNγ released in response to 1 nM IL-18 (+1.1 nM TNFα) wasmonitored in each experiment, and ranged from approximately 250 to 4000pg/ml.

The KG-1 assay was carried out in 96 flat bottom well cell cultureplates (Costar). Test solutions of antibody (in duplicate) were usedneat (or diluted to the required concentration in Dulbecco's PBS) in avolume of 50 The antibodies were then titrated in a 6-9 point ⅓ dilutionseries (using KG-1 medium), followed by the addition of 50 μl KG-1 cellswith gentle mixing. A “no antibody” control with only IL-18 and a “cellsonly” control were always included. After incubation of theantibody/cell mixture for 30-60 minutes at 37° C. with 5% CO₂, 100 μl ofIL-18+TNFα diluted in KG-1 medium was added with gentle mixing. Thefinal concentration of IMAC-purified scFv typically ranged between25-200 μg/ml. Three reference inhibitors were used in all assays. Thefirst two were monoclonal antibodies against the two different chains ofthe IL-18R, RP1 (R&Dsystems, MAB840) and AcPL (R&D Systems, MAB1181).These antibodies were used as described above for scFv, except that thefinal starting concentration for the dilution series was 10-20 μg/ml. Inaddition, a recombinant IL-18BP/Fc chimera (R&D Systems, 119-BP) wasused to neutralize IL-18. In this case, 50 μl of KG-1 medium was addedto the cell culture plate, followed by 50 μl of cells and the cells wereincubated as above for the antibodies. In a separate 96 well “U” bottomcell culture plate, the IL-18BP/Fc was titrated in a 6-9 point ½dilution series (using KG-1 medium) in a volume of 60 μl/well. An equalvolume of IL-18+TNFα was added to the IL-18BP/Fc dilution series. Afterincubation of the IL-18BP/Fc/IL-18 mixture for 30-60 minutes at 37° C.with 5% CO₂, 100 μl/well was added to the KG-1 cell plate with gentlemixing. The final starting concentration of the IL-18BP/Fc for thedilution series was 1 mg/ml. The final concentration of the KG-1 cellswas 1.5×10⁶ cells/ml (i.e. 3×10⁵/well) and IL-18 was used at a finalconcentration of 1 nM (+1.1 nM TNFα).

IL-18 was also titrated to determine the EC50 of the IFNγ response. TheIL-18 was titrated in a 6-10 point ½ dilution series (using KG-1medium), with the TNFα held constant at 1.1 nM. In this case 50 ml ofKG-1 medium was added to the cell culture plate, followed by 50 ml ofcells. The cell plate was incubated for 30-60 minutes at 37° C. with 5%CO₂, then 100 ml of the titrated IL-18 was added with gentle mixing. Thefinal concentration of IL-18 started at 5 nM for the dilution series.Typically, the EC₅₀ for IL-18 (+TNFα) was in the range of 0.5-1 nM,although minimum and maximum EC₅₀ values of down to 0.3 nM or up to 5 nMwere occasionally seen.

The KG-1 cell plates were incubated at 37° C. with 5% CO₂ overnight. Thecell supernatants were harvested by removal of 180 ml medium into aclean “U” bottom 96-well plate, which was then sealed and centrifuged at1200 rpm for 5 minutes. 160 ml of the cell free supernatant was thentransferred to another clean “U” bottom 96-well plate, and the clarifiedsupernatants tested immediately or frozen at −20° C.

The amount of IFNγ in the KG-1 cell supernatants was determined by astandard sandwich ELISA based assay, using a time-resolved fluorometricreadout. FLUORONUNC flat bottom 96-well plates (NUNC, 437958) werecoated using 100 ml/well of the IFNγ specific monoclonal captureantibody (R&D Systems, MAB2851) at 4 mg/ml and left at +4° C. overnight.The plates were rinsed with Dulbecco's PBS, then non-specific proteinbinding was blocked by the addition of 200 ml/well of 3% milk powder inPBS and incubation at RT (RT) for 1-2 hours. The recombinant human IFNγstandard (R&D Systems, 285-IF-100) was diluted to 16,000 pg/ml inreagent diluent (0.1% BSA, 0.05% Tween-20, 20 mM Tris, 150 mM NaCl; pH7.2-7.4), then titrated in a 12 point ½ dilution series. The blockingbuffer was removed from the capture antibody coated plates, and 100ml/well of the IFNγ standard or clarified cell supernatants was added.Reagent diluent was added for the “blank” control. After incubation for1-2 hours at RT, the plates were washed 3× with PBS containing 0.1%Tween-20. The biotinylated anti-human IFNγ polyclonal detection antibody(R&D Systems, BAF285) was diluted to 100 ng/ml in reagent diluentsupplemented with 2% normal goat serum, and 100 ml/well was added. Afterincubation for 1 hour at RT, the plates were washed 3× with PBScontaining 0.1% Tween-20. Streptavidin-Europium (Perkin-Elmer,4001-0010) was diluted 1/1000 in DELFIA assay buffer (Perkin-Elmer,4002-0010), and 100 ml/well was added. After 30-60 minutes incubation atRT, the plates were washed 7× with DELFIA wash buffer (Perkin-Elmer,1244-114). DELFIA Enhancement solution (Perkin-Elmer, 4001-0010) wasadded at 100 ml/well and the plates were left for at least 10 min at RT.The resulting fluorescent signal was measured usingdissociation-enhanced time-resolved fluorometryusing the Victor2 V platereader (PerkinElmer).

The average value for the ELISA “blank” control was subtracted from theresults for the IFNγ standard, while the average value for the “cellsalone” control was subtracted for the cell supernatant results. GraphPadPRISM (GraphPad Software, Inc.) was used to calculate the IFNγ standardcurve using non-linear regression (with a variable slope). Theconcentration of IFNγ in the cell supernatants was then determined byusing an “unknown X from Y” output for the IFNγ standard curve. The EC50for IFNγ release from KG-1 cells in response to IL-18 was calculatedusing non-linear regression (with a variable slope) and constraining thetop and bottom as necessary. Inhibition of IFNγ release from KG-1 cellsby test compound was normalised as a percentage of the average value ofthe “no antibody” IL-18 alone control, using the fluorescent countsdata. The IC₅₀ values for test compounds could then be calculated usingnon-linear regression (with a variable slope), and constraining thebottom and top to 0 and 100% respectively.

The IgG2 versions of antibodies AM_(H)9/AM_(L)9, AM_(H)11/AM_(L)7,AM_(H)3/AM_(L)14, and AM_(H)6/AM_(L)12 have at least equivalent potencyas the original IgG4 versions in an assay measuring their effect on theIFN-γ production by KG1 cells. Furthermore, the IgG2 versions have atleast equivalent potency as the original IgG4s in an assay measuringINF-γ production by human NK cells.

The IgG2 versions of the antibodies have equivalent potency as theoriginal IgG4s in an assay measuring their effect on IL-18 induced INF-γproduction by cynomolgus PBMC#010182 cells. This confirms that theconversion to IgG2 did not affect the cyno cross reactivity of thetested antibodies.

4. Assays for Specificity

IgGs of various antibodies were analyzed for cross-reactivity by ELISAagainst a panel of proteins.

Test antigens were coated onto Protein Immobiliser 96-well plates(Exiqon, Prd#10203-111-60) at 1 μg/ml in PBS (Dulbecco's w/o Ca and Mg,Invitrogen, Cat#14190-086) in duplicate, 50 μl per well, overnight at 4°C.

Plates were washed three times with 300 μl PBS-Tween (0.1%) (PBS-T) andthree times with 300 μl PBS per well using a 96-well plate washer(BIO-TEK, ELX405UV). To the washed plates, 300 μl of 3% Marvel PBS(MPBS) was added per well as a blocking agent. Plates were blocked atroom temperature (RT) for 1 hour.

Plates were washed three times with PBS-T and three times with PBS aspreviously stated. Antibodies (hulgG₄) were diluted to 0.5 μg/ml in 3%MPBS. 50 μl of diluted hulgG₄ were added per well. Plates were incubatedat RT for 1 hour. Plates were washed as previously stated.

Primary detection antibody (Monoclonal anti-human IgG4 clone HP-6025biotin conjugate, Sigma, Cat#B-3648) was diluted 1:15,000 in 3% MPBS andadded to plates at 50 μl per well. Incubation with primary detectionantibody was at RT for 1 hour. Plates were washed previously stated.

Secondary detection antibody (ExtrAvidin peroxidase conjugate, Sigma,Cat#E-2886), was diluted 1:1,000 in 3% MPBS and added to plates at 50 μlper well. Incubation with secondary detection antibody was at RT for 30minutes. Plates were washed previously stated.

50 μl per well Tetramethyl-benzidine (TMB) (Liquid substrate for ELISA,Sigma, Cat#T-0440) was added and incubated at RT for 10 minutes. To stopthe enzyme color reaction, 50 μl 0.5 M H₂SO₄ per well was added.

Plates were read at 450 nm on a 96-well plate reader (Victor² V platereader (PerkinElmer).

The specific anti-IL-18 receptor IgG4 antibodies were positive againsthuman and cynomolgus IL-18 receptor protein only. There was nocross-reactivity with other species. An IgG2 version of an aboveantibody had the same cross-reactivity properties, i.e., itcross-reacted with cynomolgus IL-18 receptor only.

B. Example 2 Characterization of the Binding Affinity of an IL-18Receptor Antibody

This Example provides an exemplary method of determining the bindingaffinity of an IL-18 receptor antigen binding protein to cellsurface-expressed human IL-18Rα. An IL-18 receptor antibody wasiodinated using [¹²⁵I]-Bolton-Hunter Reagent (diiodinated; PerkinElmerLife Sciences, Inc., Boston, Mass.). One millicurie of[¹²⁵I]-Bolton-Hunter Reagent supplied in anhydrous benzene wasevaporated to dryness under a nitrogen stream. Five microliters of IL-18receptor antibody (16 micrograms) was diluted with an equal volume ofborate buffered saline and then added to the dried [¹²⁵1]-Bolton-HunterReagent in its original vial. After an overnight incubation at 4° C., 10microliters of PBS, 0.1% gelatin was added and the entire sampletransferred to an equilibrated 2 mL P6 column (BioRad; Hercules, Calif.)where iodinated IL-18 receptor antibody was separated from free ¹²⁵I bygel filtration with 0.1% gelatin as a carrier protein. Fractionscontaining iodinated antibody were pooled, diluted to a concentration of100 nM in binding media (RPMI 1640 containing 2.5% bovine albumin,Fraction V, 20 mM Hepes, and 0.2% sodium azide, pH 7.2), and stored at4° C. A specific activity of 3.5×10¹⁵ cpm/mmol was calculated based onan initial protein concentration of antibody by amino acid analysis, anda recovery of 70% from a control experiment in which an aliquot ofiodiniated antibody was put through the iodination protocol withomission of [¹²⁵I]-Bolton-Hunter Reagent.

1. Direct Equilibrium Binding

KG-1 cells were stimulated for 20 hours at 37° C. in 5% CO₂ in IMDMmedium containing 20% fetal calf serum in the presence of 20 ng/mL ofhuman TNFα. The stimulated KG-1 cells (5×10⁵ cells/150 microlitersfinal) were washed twice with PBS, and then incubated with a range ofconcentrations of iodinated antibody. Nonspecific binding was measuredat a single concentration of iodinated antibody (˜350 pM, in triplicate)in the presence of a 1000-fold molar excess of unlabeled antibody, andassumed to be a linear function of the concentration of iodinatedantibody present. All reagents were diluted in binding media containingsodium azide (0.2%) to inhibit potential internalization of iodinatedantibody by the cells.

Cells were incubated in 96-well round-bottom microtiter plates at 37°C., 5% CO₂ on a miniorbital shaker. After 4 hours, two 60 microliteraliquots of each mixture were transferred to chilled 400 microliterpolyethylene centrifuge tubes containing 200 microliters phthalate oil(1.5 parts dibutylphthalate: 1 part dioctylphthalate) and spun for 1.5minutes in a 4° C. tabletop microfuge (Sorvall, Asheville, N.C.) at10,000 rpm to separate cell associated iodinated antibody from freeiodinated antibody. The oil tubes were cut, and each cell pellet andsupernatant was collected in individual 12×75 mm glass tubes and loadedon a COBRA gamma counter (Packard Instrument Company; Boston, Mass.) forcpm measurements. Cpm from duplicate aliquots for each well wereaveraged for analysis. Data were fir to a simple 1-site binding equationvia nonlinear regression in Prism version 3.03 (GraphPad Software, Inc;San Diego, Calif.).

The iodinated antibody bound to stimulated KG-1 cells at 37° C. with aK_(D) of 81 pM and ˜4700 sites/cell.

2. Competition Assay

Stimulated and washed KG-1 cells (5×10⁵ cells/150 microliters final)were incubated with a single concentration of iodinated antibody (15.6pM) and varying concentrations of unlabeled antibody in binding media.Nonspecific binding was determined in the presence of a 1000-fold molarexcess of unlabeled antibody. Iodinated and unlabeled antibody weremixed just prior to the addition of cells, i.e., there was nopre-incubation step.

Cells were incubated in 96-well round-bottom microtiter plates at 37°C., 5% CO₂ on a miniorbital shaker. After 4 hours, two 60 microliteraliquots of each mixture were transferred to chilled 400 microliterpolyethylene centrifuge tubes containing 200 microliters phthalate oil(1.5 parts dibutylphthalate: 1 part dioctylphthalate) and spun for 1.5minutes in a 4° C. tabletop microfuge (Sorvall, Asheville, N.C.) at10,000 rpm to separate cell associated iodinated antibody from freeiodinated antibody. The oil tubes were cut, and each cell pellet andsupernatant was collected in individual 12×75 mm glass tubes and loadedon a COBRA gamma counter (Packard Instrument Company; Boston, Mass.) forcpm measurements. Cpm from duplicate aliquots for each well wereaveraged for analysis. Data were fit to a single competitive inhibitionequation via nonlinear regression using the Kd value of 81 pM in Prism.

The K_(I) of unlabeled antibody binding was 53 pM.

C. Example 3 Characterization of the Activity of Anti-IL-18 ReceptorAntibodies

A IFNγ release assay was performed as described in Example 1, Section 3for various IgG constructs, as described below.

1. Comparison of the Inhibition of INFγ Release by KG1 Cells withAM_(H)2/AM_(L)16, AM_(H)2/AM_(L)17, AM_(H)1/AM_(L)16, andAM_(H)1/AM_(L)17 Constructs

Inhibition of INF-γ release by KG1 cells when treated with AM_(H)/AM_(L)antigen binding protein constructs AM_(H)2/AM_(L)16, AM_(H)2/AM_(L)17,AM_(H)1/AM_(L)16, and AM_(H)1/AM_(L)17 was compared with controltreatment with IL-18 binding protein (BP). The AM_(H)/AM_(L) antigenbinding proteins were significantly more efficacious at inhibiting IFNγrelease, all demonstrating an ED₅₀ between 6 and 10 pM compared to anED₅₀ for IL-18 BP of 520 pM.

2. Comparison of the Inhibition of INFγ Release by KG1 Cells withAM_(H)4/AM_(L)14, AM_(H)4/AM_(L)15, AM_(H)3/AM_(L)14, andAM_(H)3/AM_(L)15 Constructs

Inhibition of INF-γ release by KG1 cells when treated with AM_(H)/AM_(L)antigen binding protein constructs AM_(H)4/AM_(L)14, AM_(H)4/AM_(L)15,AM_(H)3/AM_(L)14, and AM_(H)3/AM_(L)15 was compared with controltreatment with IL-18 binding protein (BP). The AM_(H)/AM_(L) antigenbinding proteins were significantly more efficacious at inhibiting IFNγrelease, all demonstrating an ED₅₀ between 3 and 7 pM compared to anED₅₀ for IL-18 BP of 520 pM.

3. Comparison Of The Inhibition Of INFγ Release by KG1 Cells withAM_(H)6/AM_(L)12, AM_(H)6/AM_(L)13, AM_(H)5/AM_(L)12, andAM_(H)5/AM_(L)13 Constructs

Inhibition of INF-γ release by KG1 cells when treated with AM_(H)/AM_(L)antigen binding protein constructs AM_(H)6/AM_(L)12, AM_(H)6/AM_(L)13,AM_(H)5/AM_(L)12, and AM_(H)5/AM_(L)13 was compared with controltreatment with IL-18 binding protein (BP). The AM_(H)/AM_(L) antigenbinding proteins were significantly more efficacious at inhibiting IFNγrelease, all demonstrating an ED₅₀ between 2.9 and 11.3 pM compared toan ED₅₀ for IL-18 BP of 520 pM.

4. Inhibition Of INFγ Release by KG1 Cells by AM_(H)8/AM_(L)11,AM_(H)9/AM_(L)9, AM_(H)10/AM_(L)8, and AM_(H)11/AM_(L)7 IgGs

Inhibition of INF-γ release by KG1 cells with AM_(H)/AM_(L) IgG antigenbinding protein constructs comprising combinations of AM_(H)8/AM_(L)11,AM_(H)9/AM_(L)19, AM_(H)10/AM_(L)8, and AM_(H)11/AM_(L)7 IgGs wascompared with control treatment with IL-18 binding protein (BP). TheAM_(H)/AM_(L) antigen binding proteins were significantly moreefficacious at inhibiting IFNγ release, all demonstrating an ED₅₀between 3 and 45 pM compared to an ED₅₀ for IL-18 BP of 520 pM.

5. Inhibition Of INFγ Release by KG1 Cells by AM_(H)15/AM_(L)3,AM_(H)13/AM_(L)4, AM_(H)13/AM_(L)5, and AM_(H)16/AM_(L)2 IgGs

Inhibition of INF-γ release by KG1 cells with AM_(H)/AM_(L) IgG antigenbinding protein constructs comprising combinations of AM_(H)15/AM_(L)3,AM_(H)13/AM_(L)4, AM_(H)13/AM_(L)5, and AM_(H)16/AM_(L)2 IgGs wascompared with control treatment with IL-18 binding protein (BP). TheAM_(H)/AM_(L) antigen binding proteins were significantly moreefficacious at inhibiting IFNγ release, all demonstrating an ED₅₀between 17 and 320 pM compared to an ED₅₀ for IL-18 BP of 520 pM.

D. Example 4 Identification of the Binding Epitope of the DescribedIL-18 Receptor Antibodies

Experiments were carried out to determine the amino acids residuespresent in human IL-18 receptor (IL-18R) that are important for bindingto one or more of the IL-18 receptor binding proteins. An exemplaryantibody bound with high affinity to human IL-18R but did not bind themouse ortholog of IL-18R. Experiments were directed towards examiningthe amino acids that differ between human and mouse IL-18R. This wascoupled with analysis of a three dimensional computational model of theIL-18R to determine which of those amino acids lie on the surface of thereceptor and are therefore more likely to interact with the antibody.Candidate amino acids were examined for their contribution to antibodyrecognition by using site-specific mutagenesis to change the particularamino acids from the human sequence to mouse sequence and then testingthe mutant IL-18R molecules for binding to antibody. The relativeability of antibody to bind the different mutations was assessed usingantibody dose-response curves and subsequent determination of EC₅₀concentrations (the concentration of antibody required for 50% of themaximal binding signal).

A region on the surface of human IL-18R was identified that isparticularly important for antibody binding and thus contributes to theepitope. This region contains amino acids 243-271 (shown in bold in FIG.5). When specific amino acids in this region were mutated to mousesequence, antibody binding was diminished (effects on antibody bindingreported in TABLE 3). Amino acids 250-253 (MFGE mutant) and 267-271(MRIMT mutation) had the most profound influence on antibody bindinghowever neither completely influenced antibody binding alone (see,underlined amino acid residues). When the receptor was mutated at allfour of the specific sites tested, antibody binding was virtuallyabolished. Binding of a control anti-IL-18R receptor antibody was notsignificantly affected by the mutations indicating that the overallstructure of the receptor had not been disrupted by the mutations. Aminoacids 243-271 encode one of the predicted IL-18 contact points and thusthis epitope is consistent with the antibody's ability to block IL-18binding to the receptor.

TABLE 3 Results of Antibody Binding Assay with IL-8R and Mutated IL-18R(the mouse amino acids corresponding to each mutation is provided inparenthesis) Fold decrease Exemplary Ab in binding Control Ab binding:ability relative binding: avg. EC50 (pM) to huIL18R avg. EC50 (pM)huIL18R 15.7 +/− 9.0 0  7.9 +/− 4.7 EE_(D)V mutation  25.3 +/− 11.9 1.6 6.7 +/− 4.4 (KDDL) MFGE mutation  57.9 +/− 26.7 3.7  5.9 +/− 3.7 (SIRK)MRIMT mutation 177.5 +/− 58.9 11.3  8.5 +/− 5.1 (TTTWI) STGGT (NEEAI)15.7 +/− 8.5 0 10.0 +/− 6.2 EE_(D)V + MRIMT + 2615.1 2287.3  7.9 +/− 5.1STGGT EE_(D)V + MRIMT + too low to N/A 10.3 +/− 5.5 MFGE + STGGT measureMouse IL18R does not bind N/A N/A

Human IL18R was mutated to mouse IL18R sequence at the indicatedresidues and recombinant mutated receptors with and avidin tag wereimmobilized on biotin-coated beads. Immobilized receptor was then usedto determine relative antibody binding ability in a soluble bindingassay. Binding was also performed using an anti-huIL-18R antibody thatrecognizes a distinct epitope from the exemplary antibody. Bindingexperiments were all performed at least two times. The average EC50represents the concentration of antibody required to achieve 50% ofmaximal binding.

What is claimed is:
 1. A method for treating a condition associated withIL-18 receptor in a patient, comprising administering to a patient inneed thereof a pharmaceutical composition comprising an antigen bindingprotein that binds the IL-18 receptor, wherein said antigen bindingprotein comprises: an antibody heavy chain comprising (a) a CDRH1 of SEQID NO:104; (b) a CDRH2 of SEQ ID NO:105; and (c) a CDRH3 of SEQ IDNO:106; and an antibody light chain comprising (a) a CDRL1 of SEQ IDNO:173; (b) a CDRL2 of SEQ ID NO:174; and (c) a CDRL3 of SEQ ID NO:175.2. The method of claim 1, wherein the antibody heavy chain comprises SEQID NO:6.
 3. The method of claim 2, wherein the antibody heavy chaincomprises SEQ ID NO:73.
 4. The method of claim 1, wherein the antibodylight chain comprises SEQ ID NO:29.
 5. The method of claim 4, whereinthe antibody light chain comprises SEQ ID NO:75.
 6. The method of claim1, wherein the antibody heavy chain comprises SEQ ID NO:6 and theantibody light chain comprises SEQ ID NO:29.
 7. The method of claim 1,wherein the antibody heavy chain comprises SEQ ID NO:73 and the antibodylight chain comprises SEQ ID NO:75.
 8. The method of claim 1 wherein thecondition is selected from the group consisting of an autoimmunedisease, a hepatic disease, a pancreatic disease, pulmonary disease, anda cardiovascular disease.
 9. The method of claim 8, wherein saidautoimmune disease is selected from the group consisting of psoriasis,rheumatoid arthritis, lupus, Type I diabetes, Crohn's disease,inflammatory bowel disease, multiple sclerosis, autoimmune hepatitis,atopic dermatitis, myasthenia gravis, and sarcoidosis.
 10. The method ofclaim 8, wherein said hepatic disease is hepatitis.
 11. The method ofclaim 8, wherein said pancreatic disease is chronic pancreatitis oracute pancreatitis.
 12. The method of claim 8, wherein saidcardiovascular disease is selected from the group consisting of heartdisease including acute heart attacks, atheromatous plaque rupture,post-ischemic heart failure, reperfusion injury, vascular inflammation,and atherogenesis.
 13. The method of claim 8, wherein the pulmonarydisease is selected from the group consisting of chronic obstructedpulmonary disease, chronic severe asthma, and acute respiratory distresssyndrome.
 14. A method of inhibiting the binding of IL-18 to IL-18receptor comprising contacting an IL-18 receptor with an antigen bindingprotein comprising: an antibody heavy chain comprising (a) a CDRH1 ofSEQ ID NO:104; (b) a CDRH2 of SEQ ID NO:105; and (c) a CDRH3 of SEQ IDNO:106; and an antibody light chain comprising (a) a CDRL1 of SEQ IDNO:173; (b) a CDRL2 of SEQ ID NO:174; and (c) a CDRL3 of SEQ ID NO:175,wherein said antigen binding protein binds said IL-18 receptor andprevents binding of said IL-18 receptor to IL-18.